JP7028807B2 - Genome assembly and haplotype fading methods - Google Patents

Description

Mutual Reference to Related Applications This application is the benefit of U.S. Patent Application No. 61 / 759,941 filed February 1, 2013 and U.S. Patent Application No. 61 / 892,355 filed October 17, 2013. Is claimed, the disclosure of which is incorporated herein by reference.

The present disclosure provides methods of genomic assembly and haplotype fading for identifying short, medium and long term connections within the genome.

Producing high-quality, highly contiguous genomic sequences remains theoretically and practically difficult.

A persistent drawback of next-generation sequencing (NGS) data is the inability to span large repeat regions of the genome due to short read lengths and relatively small insertion sizes. This flaw significantly affects the de novo assembly. Contigs separated by long repeat regions cannot be linked or rearranged due to the uncertain nature and arrangement of genomic rearrangements. Moreover, fading information cannot be determined because variants cannot be clearly associated with haplotypes over long distances. The present disclosure solves all of these problems simultaneously by generating a very long range of read pairs (XLRP) over a genomic distance of approximately hundreds of kilobases and the longest megabase using appropriate input DNA. Can be done. Such data can be invaluable to overcome the substantial barriers posed by large repeat regions in the genome, including centromeres, enabling cost-effective de novo assembly. Sufficient integrity and accuracy of rearrangement determination data can be produced for personalized medicine.

Of significant importance is the use of reconstituted chromatin in the formation of associations between DNA segments that are very distant but molecularly linked. The present disclosure allows distant segments to come together and be covalently linked by chromatin higher order structures, thereby physically connecting previously distant portions of a DNA molecule. Subsequent processing allows the sequence of associated segments to be verified and allows segregation on the genome to produce read pairs that extend to the full length of the input DNA molecule. Since the lead pairs are derived from the same molecule, these pairs also contain phase information.

In some embodiments, the present disclosure provides a method by which a high quality assembly can be made with much less data than previously required. For example, the method disclosed herein provides a genomic assembly from data from only two lanes of Illumina HiSeq.

In other embodiments, the present disclosure provides a method capable of generating chromosomal level fading using a long-range read pair technique. For example, the methods disclosed herein are capable of fading 90% or more of heterozygous single nucleotide polymorphisms (SNPs), each with at least 99% accuracy. This accuracy is substantially higher cost and is comparable to fading produced by a time-consuming and labor-intensive method.

In some examples, methods capable of producing fragments of genomic DNA up to the megabase scale can be used in conjunction with the methods disclosed herein. It is possible to confirm the ability of this method to generate long DNA fragments and generate read pairs across the longest fragments provided by their extraction. In some cases, DNA fragments larger than 150 kbp in length can be extracted and used to generate XLRP libraries.

The present disclosure provides a way to significantly accelerate and improve the de novo genome assembly. The methods disclosed herein utilize data analysis methods that allow rapid and inexpensive de novo assembly of genomes from one or more subjects. The disclosure further provides that the methods disclosed herein can be used for a variety of applications, including haplotype fading and metagenomic analysis.

In certain embodiments, the present disclosure comprises generating multiple contigs and generating multiple read pairs from data produced by probing the physical layout of a chromosome, chromatin or reconstituted chromatin. The step of mapping or assembling multiple read pairs to multiple contigs, the step of constructing adjacent contig matrices using read mapping or assembly data, and the analysis of the adjacent matrices and their order and / or direction with respect to the genome. Provided is a method of genome assembly including a step of determining a pathway through a contig, which represents. In a further embodiment, the present disclosure weights at least about 90% of lead pairs by obtaining a mapping of the distance of each lead to the end of the contig, which leads show short range of contact and which leads. It provides to incorporate information about whether a pair exhibits a longer range of contact. In other embodiments, the adjacency matrix is rescaled to represent indiscriminate regions of the genome, such as conservative binding sites for one or more agents that regulate chromatin scaffold interactions, such as transcriptional repressor CTCF. The weight of many contacts on the contig can be reduced. In other embodiments, the present disclosure provides a method of genomic assembly of a human subject, whereby multiple contigs are generated from the DNA of the human subject, thereby making a chromosome of the human subject from the naked DNA of the subject. Multiple lead pairs are generated by analyzing chromatin or reconstituted chromatin.

In a further embodiment, the present disclosure comprises the steps of fragmenting a long stretch of DNA of interest into random pieces of uncertain size and sequencing the pieces using a high-throughput sequencing method. It provides that multiple contigs can be generated by using a shotgun sequencing method that includes a step of generating an sequencing read and a step of assembling the sequencing read to form multiple contigs.

In certain embodiments, the present disclosure provides that multiple read pairs can be generated by probing the physical layout of a chromosome, chromatin or reconstituted chromatin using Hi-C based techniques. In a further embodiment, the Hi-C based technique involves cross-linking a chromosome, chromatin or reconstituted chromatin with a fixative such as formaldehyde to form a DNA-protein bridge, and cross-linking with one or more restriction enzymes. The step of cleaving the DNA-protein to form multiple DNA-protein complexes containing sticky ends, and filling the sticky ends with nucleotides containing one or more markers, such as biotin, and then ligating together. High-throughput sequencing, including the steps to create blunt ends, fragmenting multiple DNA-protein complexes into fragments, and pulling down junctions containing fragments by using one or more markers. Includes the steps of sequencing the junction containing the fragment using the to generate multiple read pairs. In a further embodiment, the plurality of lead pairs for the methods disclosed herein are generated from the data produced by probing the physical layout of the reconstituted chromatin.

In various embodiments, the present disclosure provides that multiple read pairs can be determined by probing the physical layout of a chromosome or chromatin isolated from cultured cells or primary tissue. In another embodiment, multiple read pairs probe the physical layout of reconstituted chromatin formed by complexing naked DNA from one or more target samples with isolated histones. It can be decided by.

In another embodiment, the disclosure provides a method of determining haplotype fading, comprising identifying one or more heterozygous sites in multiple read pairs, the pair of heterozygous sites. Fading data for allelic variants can be determined by identifying the included read pairs.

In various embodiments, the present disclosure is modified to include a modified step of collecting microorganisms from the environment and a modified step of adding a fixing agent such as formaldehyde to form a contig within each microbial cell. Which contigs are the same lead pairs that are mapped to different contigs, including the step of generating multiple read pairs by probing the physical layout of multiple microbial chromosomes using a Hi-C based method. Provided is a method of high throughput bacterial genome assembly to indicate whether it is derived from a species.

In some embodiments, the disclosure presents a plurality of read pairs from data generated by (a) stepping to generate multiple contigs and (b) probing the physical layout of a chromosome, chromatin or reconstituted chromatin. Steps to determine, (c) map multiple read pairs to multiple contigs, (d) build contig adjacencies using read mapping data, and (e) analyze adjacency matrices. A method of genome assembly comprising the steps of determining the pathway through the contig, which represents its order and / or direction with respect to the genome, is provided.

In a further embodiment, the present disclosure provides a method of generating multiple read pairs by probing the physical layout of a chromosome, chromatin or reconstituted chromatin using Hi-C based techniques. In a further embodiment, the Hi-C based technique involves (a) cross-linking a chromosome, chromatin or reconstituted chromatin with a fixative to form a DNA-protein bridge, and (b) one or more restriction. Steps to cleave the enzymatically cross-linked DNA-protein to form multiple DNA-protein complexes containing sticky ends, and (c) fill the sticky ends with nucleotides containing one or more markers, and then Steps to create blunt ends to be ligated together, (d) to shear multiple DNA-protein complexes into fragments, and (e) junctions containing the fragments by using one or more markers. It includes a pull-down step and (f) sequencing the junction containing the fragment using a high throughput sequencing method to generate multiple read pairs.

In certain embodiments, multiple read pairs are determined by probing the physical layout of chromosomes or chromatin isolated from cultured cells or primary tissue. In another embodiment, multiple read pairs probe the physical layout of reconstituted chromatin formed by complexing naked DNA from one or more target samples with isolated histones. It is determined by that.

In some embodiments, at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99% or by obtaining a mapping of the lead distance to the end of the contig. Weight more multiple lead pairs to incorporate a higher probability of short contacts than long contacts. In some embodiments, the adjacency matrix is rescaled to reduce the weight of many contacts on some contigs that represent indiscriminate regions of the genome.

In certain embodiments, the indiscriminate region of the genome comprises one or more conservative binding sites for one or more agents that regulate chromatin scaffold interactions. In some examples, the agent is transcriptional repressor CTCF.

In some embodiments, the methods disclosed herein provide a genomic assembly of a human subject, whereby multiple contigs are generated from the DNA of the human subject, thereby making a human from the naked DNA of the subject. Multiple read pairs are generated from analysis of the chromosome, chromatin or reconstituted chromatin of interest.

In another embodiment, the disclosure provides a method of determining haplotype fading comprising identifying one or more heterozygous sites in multiple read pairs, comprising a pair of heterozygous sites. By identifying lead pairs, fading data for allelic variants can be determined.

In yet another embodiment, the disclosure provides a method of metagenomic assembly in which multiple lead pairs form a crosslink within each microbial cell by adding a fixative and a step of collecting the microorganism from the environment. Lead pairs generated by probing the physical layout of multiple microbial chromosomes using a modified Hi-C-based method, including steps to, and which contigs are of the same species. Indicates whether it is derived. In some examples, the fixative is formaldehyde.

In some embodiments, the present disclosure is derived from a single DNA molecule, comprising the step of generating multiple read pairs from a single DNA molecule and the step of assembling contigs using read pairs. It provides a way to assemble contigs, with at least 1% of read pairs spanning distances greater than 50 kB on a single DNA molecule, and read pairs are generated within 14 days. In some embodiments, at least 10% of read pairs span distances greater than 50 kB on a single DNA molecule. In other embodiments, at least 1% of read pairs span a distance greater than 100 kB on a single DNA molecule. In a further embodiment, lead pairs are generated within 7 days.

In other embodiments, the present disclosure derives from a single DNA molecule, comprising the step of generating multiple read pairs from a single DNA molecule in vitro and the step of assembling contigs using read pairs. It provides a way to assemble multiple contigs, with at least 1% of read pairs spanning distances greater than 30 kB on a single DNA molecule. In some embodiments, at least 10% of read pairs span distances greater than 30 kB on a single DNA molecule. In other embodiments, at least 1% of read pairs span a distance greater than 50 kB on a single DNA molecule.

In yet another embodiment, the disclosure comprises a method of haplotype fading comprising the steps of generating multiple read pairs from a single DNA molecule and assembling multiple contigs of the DNA molecule using the read pairs. With at least 1% of read pairs spanning distances greater than 50 kB on a single DNA molecule, haplotype fading is performed with an accuracy of over 70%. In some embodiments, at least 10% of read pairs span distances greater than 50 kB on a single DNA molecule. In other embodiments, at least 1% of read pairs span a distance greater than 100 kB on a single DNA molecule. In a further embodiment, haplotype fading is performed with an accuracy greater than 90%.

In a further embodiment, the disclosure comprises haplotype fading in vitro to generate multiple read pairs from a single DNA molecule and to assemble multiple contigs of the DNA molecule using the read pairs. With at least 1% of read pairs spanning distances greater than 30 kB on a single DNA molecule, haplotype fading is performed with an accuracy of> 70%. In some embodiments, at least 10% of read pairs span distances greater than 30 kB on a single DNA molecule. In other embodiments, at least 1% of read pairs span a distance greater than 50 kB on a single DNA molecule. In yet another embodiment, haplotype fading is performed with an accuracy greater than 90%. In a further embodiment, haplotype fading is performed with an accuracy greater than 70%.

In some embodiments, the present disclosure is (a) a step of cross-linking a first DNA molecule in vitro, wherein the first DNA molecule comprises a first DNA segment and a second DNA segment. And (b) the step of linking the first DNA segment to the second DNA segment to form the linked DNA segment, and (c) sequencing the linked DNA segment, thereby the first read. Provided is a method for generating a first read pair from a first DNA molecule, which comprises the step of obtaining a pair.

In some embodiments, multiple associated molecules, such as those derived from reconstituted chromatin, are crosslinked to a first DNA molecule. In some examples, the associated molecule comprises an amino acid. In a further example, the associated molecule is a peptide or protein. In certain embodiments, the first DNA molecule is crosslinked with a fixative. In some examples, the fixative is formaldehyde. In some embodiments, the first DNA segment and the second DNA segment are generated by cleaving the first DNA molecule. In certain embodiments, the method further comprises assembling multiple contigs of the first DNA molecule using the first read pair. In some embodiments, each of the first and second DNA segments is attached to at least one affinity label and the linked DNA segment is captured using the affinity label.

In a further embodiment, the method comprises (a) providing a plurality of associated molecules, such as those derived from reconstituted chromatin, to at least the second DNA molecule, and (b) bridging the associated molecule into the second DNA molecule. Thereby forming a second complex in vitro, (c) separating the second complex and thereby producing a third DNA segment and a fourth segment, and (d) a third. DNA segment is ligated to the fourth DNA segment to form a second linked DNA segment, and (e) the second linked DNA segment is sequenced, thereby obtaining a second read pair. Further includes steps. In some examples, less than 40% of DNA segments from DNA molecules are linked to DNA segments from any other DNA molecule. In a further example, less than 20% of the DNA segment from a DNA molecule is linked to a DNA segment from any other DNA molecule.

In other embodiments, the present disclosure provides (a) one or more DNA-binding molecules to a first DNA molecule, and (b) a step in which the one or more DNA-binding molecules bind to a predetermined sequence. The step of cross-linking the first DNA molecule in vitro, in which the first DNA molecule contains the first DNA segment and the second DNA segment, and (c) the first DNA segment is included in the second. A default, including the steps of ligating with a DNA segment, thereby forming a first ligated DNA segment, and (d) sequencing the first ligated DNA segment, thereby obtaining a first read pair. A method of generating a first read pair from a first DNA molecule containing a sequence, in which the probability that a given sequence will appear in the read pair is affected by the binding of the DNA binding molecule to the given sequence.

In some embodiments, the DNA binding molecule is a nucleic acid that can hybridize to a predetermined sequence. In some examples, the nucleic acid is RNA. In another example, the nucleic acid is DNA. In other embodiments, the DNA binding molecule is a small molecule. In some examples, small molecules bind to a predetermined sequence with a binding affinity of less than 100 μM. In a further example, the small molecule binds to a predetermined sequence with a binding affinity of less than 1 μM. In a further embodiment, the DNA binding molecule is immobilized on a surface or solid support.

In some embodiments, the probability that a default sequence will appear in a read pair is reduced. In other embodiments, the probability that a default sequence will appear in a read pair increases.

In yet another embodiment, the present disclosure provides an in vitro library containing a plurality of read pairs, each containing at least a first sequence element and a second sequence element, wherein the first and second sequence elements are simple. Derived from a single DNA molecule, at least 1% of read pairs contain first and second sequence elements that are at least 50 kB apart on a single DNA molecule.

In some embodiments, at least 10% of the read pairs contain first and second sequence elements that are at least 50 kB apart on a single DNA molecule. In other embodiments, at least 1% of read pairs contain first and second sequence elements that are at least 100 kB apart on a single DNA molecule.

In a further embodiment, less than 20% of read pairs contain one or more default sequences. In a further embodiment, less than 10% of read pairs contain one or more default sequences. In yet another embodiment, less than 5% of read pairs contain one or more default sequences.

In some embodiments, the default sequence is determined by one or more nucleic acids that can hybridize to the default sequence. In some examples, one or more nucleic acids are RNA. In another example, one or more nucleic acids is DNA. In a further example, one or more nucleic acids are immobilized on a surface or solid support.

In other embodiments, the default sequence is determined by one or more small molecules. In some examples, one or more small molecules bind to a predetermined sequence with a binding affinity of less than 100 μM. In a further example, one or more small molecules bind to a predetermined sequence with a binding affinity of less than 1 μM.

In some embodiments, the present disclosure provides a composition comprising a plurality of associated molecules, such as those derived from DNA fragments and reconstituted chromatin, where (a) the associated molecules are crosslinked into DNA fragments in an in vitro complex. (B) The in vitro complex is immobilized on a solid support.

In other embodiments, the present disclosure provides a composition comprising a DNA fragment, a plurality of associated molecules and a DNA binding molecule, wherein (a) the DNA binding molecule is bound to a predetermined sequence of the DNA fragment (. b) The associated molecule is cross-linked into a DNA fragment.

In some embodiments, the DNA binding molecule is a nucleic acid that can hybridize to a predetermined sequence. In some examples, the nucleic acid is RNA. In another example, the nucleic acid is DNA. In a further example, the nucleic acid is immobilized on a surface or solid support.

In other embodiments, the DNA binding molecule is a small molecule. In some examples, small molecules bind to a predetermined sequence with a binding affinity of less than 100 μM. In another example, the small molecule binds to a predetermined sequence with a binding affinity of less than 1 μM.

Incorporation by Reference All publications, patents and patent applications described herein are the same as each individual publication, patent or patent application being specifically and individually indicated as incorporated by reference. To some extent, it is incorporated herein by reference. All publications, patents and patent applications described herein are incorporated herein by reference in their entirety and any references cited therein.

The novel features of this disclosure are described in detail in the appended claims. A better understanding of the features and benefits of the present disclosure will be obtained by reference to the following detailed description of exemplary embodiments in which the principles of the present disclosure are utilized, with the following drawings.

FIG. 6 illustrates an illustration of a genome assembly using a high-throughput sequencing read. Shows the genome to be assembled (top). In general, the genome has many repetitive sequences that are difficult to assemble. Random, high-throughput sequence data from the genome is collected (center) and assembled into "contigs" in unique regions of the genome (bottom). Contig assembly usually stops at many repeats. The final output is a set of thousands of contigs whose order and orientation with respect to each other is unknown. In the figure, they are arbitrarily numbered from longest to shortest. It is a figure which illustrates the procedure based on Hi-C of this disclosure. (A) Demonstrates where DNA is cross-linked and processed to produce biotinylated junction fragments for sequencing, (B-D) Contact map data on human chr14 for various restriction enzymes. offer. As shown, most contacts are localized along the chromosome. Provided are the methods of the present disclosure that use Hi-C sequence data to assist in genomic assembly. (A) Illustrated where DNA is cross-linked and processed using Hi-C based procedures, (B) Read-to-data is generated from random shotgun sequencing and assembly. It is a diagram demonstrating the location mapped to the assembled contigs, (C) showing that after filtering and weighting, an adjacency matrix can be constructed that summarizes all inter-contig read-to-data. This matrix can be rearranged to show the correct assembly path. As shown, most lead pairs can be mapped within the contig. From there, it becomes possible to know the distribution of contact distances (see, for example, Figure 6). Read pairs that map to different contigs provide data about contig adjacencies in the correct genomic assembly. It is a figure which illustrates the typical procedure of this disclosure. DNA fragments are first generated, prepared, then in vitro chromatin assembly and biotinylated, then the chromatin / DNA complex is immobilized with formaldehyde, pulled down with streptavidin beads, and then restriction digested. The chromatin / DNA complex is subjected to proteinase digestion, exonuclease digestion and shearing, followed by biotinylated dCTP and blunt-ended ligation, followed by biotinylated dCTP and sulphated GTP. Pull down with to ligate with the sequencing adapter, and finally select the DNA fragment by size and sequence. It provides an illustration of the ambiguity that occurs in genome assembly and alignment, which derives from repetitive regions in the genome. (A) It is a figure which shows that the uncertainty in concatenation is caused by the read pair which cannot be bridged to the iterative region. (B) It is a figure which shows the uncertainty of the arrangement of a segment because a read pair cannot cross the boundary of an iteration. It is a figure which illustrates the distribution of the genomic distance between the read pairs of a human XLRP library. The maximum distances that can be achieved with other technologies are shown for comparison. FIG. 5 illustrates fading accuracy for a sample using the well-characterized haplotype NA12878. The distances shown are between faded SNPs. It is a figure which illustrates various components of a typical computer system by various embodiments of this disclosure. FIG. 3 is a block diagram illustrating a typical computer system architecture that can be used with respect to the various embodiments of the present disclosure. It is a diagram illustrating a typical computer network that can be used with respect to various embodiments of the present disclosure. FIG. 3 is a block diagram illustrating another typical computer system architecture that can be used with respect to the various embodiments of the present disclosure.

As used herein and in the appended claims, the singular, "a," "and," and "the" include multiple referents unless the context clearly dictates otherwise. Thus, for example, a reference to "contig" includes the plural of such contigs, and a reference to "probing the physical layout of a chromosome" is one or more for probing the physical layout of a chromosome. Includes references to methods and their equivalents known to those of skill in the art.

The use of "and" also means "and / or" unless otherwise stated. Similarly, "comprise", "comprises", "comprising", "include", "includes", and "including" are interchangeable and are not intended to be restricted.

When the description of the various embodiments uses the term "comprising", in some specific examples the embodiments can be described instead using the phrase "consisting essentially of" or "consisting of". What one of ordinary skill in the art understands should be further understood.

As used herein, the term "sequencing read" refers to a fragment of DNA that has been sequenced.

As used herein, the term "contig" refers to a contiguous region of a DNA sequence. A "contig" compares sequencing reads to overlapping sequences and / or compares sequenced reads to a database of known sequences, with a high probability of which sequencing reads are contiguous. It can be determined by many methods known in the art, such as by identifying.

As used herein, the term "object" can refer to any eukaryote or prokaryote.

As used herein, the term "naked DNA" can refer to DNA that is substantially free of complexed proteins. For example, it is DNA that is complexed with about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, or less than about 1% of the endogenous proteins found in the cell nucleus. Can be pointed to.

As used herein, the term "reconstituted chromatin" can refer to the formation of chromatin formed by complexing an isolated nuclear protein with naked DNA.

As used herein, the term "read pair" or "read-pair" can refer to two or more elements that are concatenated to give sequence information. In some cases, the number of read pairs can refer to the number of mapable read pairs. In other cases, the number of read pairs can refer to the total number of read pairs generated.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any of the methods and reagents similar to or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, but typical methods and materials are described herein.

The present disclosure provides a method for generating a very long range of lead pairs and using that data to improve all of the work described above. In some embodiments, the present disclosure provides a method of producing a highly continuous and accurate human genome assembly with only about 3 million read pairs. In other embodiments, the present disclosure provides a method of fading 90% or more heterozygous variants in the human genome with 99% or higher accuracy. In addition, the range of read pairs generated by the present disclosure can be extended to span wider genomic distances. Assemblies are made from a standard shotgun library as well as a very long range of read-to-library. In yet another embodiment, the disclosure provides software that can utilize both of these sets of sequencing data. Faded variants are created in a single long-range read-to-library, from which reads are mapped to the reference genome, which is then used to assign the variant to one of the individual's parental chromosomes. Finally, the present disclosure provides that even larger DNA fragments are extracted using known techniques to produce very long reads.

The mechanism by which these iterations block the assembly and alignment process is quite straightforward and ultimately the result of ambiguity (Figure 5). For large repeating regions, the difficulty is one of the spans. If the lead or lead pair is not long enough to span the repeating region, the regions in contact with the repeating element cannot be clearly connected. For smaller repeating elements, the problem is primarily placement. If a region is adjacent to two repetitive elements commonly found in the genome, it is difficult, if not impossible, to determine the exact placement of the adjacent element due to its similarity to everything else in its class. become. In both cases, it is a lack of characteristic information in the iterations that makes it difficult to identify and therefore place a particular iteration. What is needed is the ability to experimentally establish connections between unique segments surrounded by or separated by repeating regions.

The methods of the present disclosure can greatly advance the field of genomics research by overcoming the substantial barriers posed by these iterative regions, thereby enabling significant advances in many domains of genomic analysis. .. To perform de novo assembly with conventional techniques, accept fragmented assemblies in many small scaffolds, or spend a lot of time and resources to create large insertion libraries or use other techniques. Must either produce a more contiguous assembly. Such techniques include obtaining very deep sequencing coverage, building BAC or phosmid libraries, optical mapping, or, in most cases, some combination of these with other techniques. obtain. Due to stringent resource and time requirements, such techniques are out of reach for most small laboratories and impede research other than model organisms. Since the method described herein can produce a very long range of read pairs, a de novo assembly can be achieved with a single sequence determination. This will reduce assembly costs by orders of magnitude and reduce the time required from months or years to weeks. In some cases, the methods disclosed herein are less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days. Allows you to generate multiple read pairs in any two ranges of less than 5 days, less than 4 days, or the time specified above. For example, the method may be able to generate multiple read pairs in about 10-14 days. Assembling the genome of any ecological status becomes routine, phylogenetic analysis will not suffer from a shortage of comparisons, and projects such as the genome 10k may become a reality.

Similarly, methods of structural and fading analysis for medical purposes remain difficult. It is surprisingly heterogeneous between cancers, individuals of the same cancer type, or even within the same tumor. Deriving the cause from the resulting effect requires very high accuracy and throughput at a low cost per sample. In the domain of personalized medicine, one of the ultimate criteria for genomic therapy was sequenced, including all fully characterized and faded variants, including large and small structural rearrangements and novel mutations. It is a genome. Achieving this with conventional technology requires similar efforts required for de novo assembly, which is currently too expensive and time consuming and labor intensive for routine medical care. The disclosed methods can rapidly generate complete and accurate genomes at low cost, thereby providing many highly explored possibilities in the study and treatment of human diseases.

Finally, by applying the methods disclosed herein to fading, the accuracy of familial analysis and the convenience of statistical methods can be combined, with more money and effort than using either method alone. And allows for sample savings. De novo variant fading, a highly desirable fading analysis that is too expensive for conventional techniques, can be readily performed using the methods disclosed herein. This is especially important because the overwhelming majority of human variants are rare (slight allele frequencies <5%). Fading information is useful for population genetic studies that benefit significantly from highly connected haplotype networks (a collection of variants assigned to a single chromosome) compared to unlinked genotypes. Haplotype information allows for higher resolution studies of historical changes in population size, migration and exchange between subpopulations, allowing specific variants to be traced back to individual parents and grandparents. When present together in a single individual, this in turn reveals the gene transduction of the variants associated with the disease and the interrelationships between the variants. The methods of the present disclosure can ultimately enable the preparation, sequencing and analysis of a very long range of read pair (XLRP) libraries.

In some embodiments of the present disclosure, tissue or DNA samples can be provided from a subject, the method of which is an assembled genome, aligned with called variants (including large structural variants), and faded variants. A call or any additional analysis can be returned. In other embodiments, the methods disclosed herein can provide the XLRP library directly to an individual.

In various embodiments of the present disclosure, the methods disclosed herein can generate a very long range of read pairs separated by a long distance. This upper limit of distance can be improved by the ability to collect large size DNA samples. In some cases, lead pairs can be up to 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 300, 400, 500, 600, 700, 800, 900, It can span 1000, 1500, 2000, 2500, 3000, 4000, 5000 kbp or longer genomic distances. In some examples, read pairs can span genomic distances up to 500 kbp. In another example, the read pair can span a genomic distance of up to 2000 kbp. The methods disclosed herein can be integrated and built onto standard techniques in molecular biology and are better suited for increased efficiency, specificity and genomic coverage. In some cases, lead pairs are about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 60 or can be produced in less than 90 days. In some examples, lead pairs can be generated in less than about 14 days. In a further example, lead pairs can be generated in less than about 10 days. In some cases, the methods of the present disclosure are at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, in correctly ordering and / or orienting multiple contigs. About 99% or about 100% accuracy, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% , About 90%, about 95%, about 99% or about 100% or more lead pairs can be provided. For example, the method can provide about 90-100% accuracy in correctly ordering and / or orienting multiple contigs.

In other embodiments, the methods disclosed herein can be used in conjunction with currently available sequencing techniques. For example, the method can be used in combination with well-tested and / or widely deployed sequencing equipment. In a further embodiment, the methods disclosed herein can be used in conjunction with techniques and techniques derived from the currently used sequencing techniques.

The methods of this disclosure dramatically simplify the de novo genome assembly for a variety of organisms. Using previous techniques, such assemblies are currently limited by short inserts in the economical mate pair library. Although it is possible to generate read pairs at genomic distances up to 40-50 kbp available in fosmid, these are expensive, cumbersome, and the longest including those in the kinematic body, which are 300 kbp-5 Mbps in size in humans. Too short to straddle the repeated stretches of. The methods disclosed herein can provide read pairs that can span long distances (eg, megabases or more), thereby overcoming these scaffold integrity challenges. .. Therefore, making chromosomal level assemblies can be routine by utilizing the methods of the present disclosure. It eliminates the need for more time-consuming and labor-intensive assembly tools that are spending tremendous time and money on current and laboratories and hindering evolving genomic catalogs, leaving resources for more meaningful analysis. You can be free. Similarly, the acquisition of long-range fading information can provide significant additional power for population genome, phylogeny, and disease research. The methods disclosed herein allow accurate fading for large numbers of individuals and thus extend the breadth and depth of the ability to probe the genome at population and deep time levels.

In the field of personalized medicine, XLRP read pairs generated from the methods disclosed herein represent significant advances for accurate, low cost, fading and rapidly produced personal genomes. Current methods lack the ability to fade variants over long distances, thereby hindering the characterization of the phenotypic effects of complex heterozygous genotypes. In addition, structural variants of substantial interest for genomic disease are accurately identified and characterized by current techniques due to their large size compared to the reads and lead-to-inserts used to study them. Is difficult. Read pairs spanning tens of kilobases to megabases or longer can help alleviate this difficulty, allowing highly parallel and individualized analysis of structural deformations.

Basic evolutionary and biomedical research is driven by technological advances in high-throughput sequencing. Whereas whole-genome sequencing and assembly is used to be the origin of large genome sequencing centers, most research universities have one or several of these machines on the market. Is currently cheap enough. Generating large amounts of DNA sequence data is currently relatively inexpensive. However, with current technology, producing high quality, highly continuous genomic sequences remains theoretically and practically difficult. Moreover, since most of the organisms we want to analyze, including humans, are diploid, each individual has two haploid copies of its genome. Which allele pair is derived from which parent at a heterozygous site (eg, the allele given by the mother is different from the allele given by the father) (known as haplotype fading). Is difficult to know. This information can be used to carry out many evolutionary and biomedical studies, including disease and trait-related studies.

In various embodiments, the present disclosure provides a method of genome assembly that combines DNA preparation techniques with paired-end sequencing to discover short, medium, and long-term connections within a given genome at high throughput. The present disclosure further provides a method of assisting genome assembly for haplotype fading and / or metagenomic studies using these connections. The method presented herein can be used to determine the assembly of the genome of interest, but the method presented herein can be used to assemble a portion of the genome of interest, such as a chromosome. Alternatively, it should be understood that the assembly of chromatin of interest of various lengths can be determined.

In some embodiments, the disclosure provides one or more methods disclosed herein comprising the step of generating multiple contigs from a sequenced fragment of target DNA obtained from a subject. Long stretches of target DNA can be fragmented by cutting the DNA with one or more restriction enzymes, shearing the DNA, or a combination thereof. The resulting fragments can be sequenced using a high throughput sequencing method to obtain multiple sequencing reads. Examples of high-throughput sequencing methods that can be used in the methods of the present disclosure are, but are not limited to, the 454 pyrosequencing method developed by Roche Diagnostics, the "cluster" sequencing method developed by Illumina, and Life Technologies. There are SOLiD and ion semiconductor sequencing methods developed by the company, and DNA nanoball sequencing methods developed by Complete Genomics. The overlapping ends of different sequencing reads can then be assembled to form a contig. Alternatively, the fragmented target DNA can be cloned into a vector. Cells or organisms are then transfected with a DNA vector to form a library. After replicating the transfected cells or organism, the vector is isolated and sequenced to generate multiple sequencing reads. The overlapping ends of different sequencing reads can then be assembled to form a contig.

As shown in Figure 1, genomic assembly can be particularly problematic in high-throughput sequencing techniques. Often, the assembly consists of thousands or even tens of thousands of short contigs. The order and orientation of these contigs is usually unknown, limiting the usefulness of genomic assembly. Techniques for ordering and orienting these scaffolds exist, but they are usually expensive, labor-intensive, and often fail to find very long-range interactions.

Samples containing target DNA used to generate contigs are collected from body fluids (eg, blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat and semen), tissue collection. It can be obtained from the subject by many means, including collecting cells / organisms. The resulting sample may be composed of a single type of cell / organism, or may be composed of multiple types of cells / organism. DNA can be extracted and prepared from the sample of interest. For example, samples can be treated with known lysis buffers, sonication techniques, electroporation, etc. to lyse cells containing polynucleotides. The target DNA can be further purified to remove contaminants such as proteins by using alcohol extraction, cesium gradient and / or column chromatography.

In another embodiment of the present disclosure, a method for extracting a very high molecular weight DNA is provided. In some cases, the data from the XLRP library can be improved by increasing the fragment size of the input DNA. In some examples, extraction of megabase-sized DNA fragments from cells can produce read pairs separated by several megabases in the genome. In some cases, the produced read pair provides sequence information over a range of about 10 kB, about 50 kB, about 100 kB, about 200 kB, about 500 kB, about 1 Mb, about 2 Mb, about 5 Mb, about 10 Mb or about 100 Mb. can do. In some examples, the read pair can provide sequence information over a range of more than about 500 kB. In a further example, the read pair can provide sequence information over a range of more than about 2 Mb. In some cases, extremely high molecular weight DNA is very mild cytolysis (Teague, B. et al., (2010) Proc. Nat. Acad. Sci. USA 107 (24), pp. 10848-53) and agarose. It can be extracted by a plug (Schwartz, DC and Cantor, CR (1984) Cell, 37 (1), pp. 67-75). In other cases, extremely high molecular weight DNA can be extracted using commercially available machines capable of purifying DNA molecules up to megabase in length.

In various embodiments, the present disclosure provides one or more methods disclosed herein comprising the steps of probing the physical layout of chromosomes in living cells. Examples of techniques for probing the physical layout of chromosomes by sequencing are chromosome higher-order structure capture (“3C”), circular chromosome higher-order structure capture (“4C”), and carbon-copy chromosome capture. ) ("5C") and "C" -based techniques such as Hi-C based methods, as well as ChIP-based methods such as ChIP-loop and ChIP-PET. These techniques utilize chromatin fixation in living cells to consolidate spatial relationships within the nucleus. Subsequent processing and sequencing of the product allows the investigator to recover the substrate of the closest association in the genomic region. Further analysis allows these associations to be used to create a three-dimensional geometric map of the chromosomes as they are physically arranged in the living nucleus. Such techniques describe the separate spatial organization of chromosomes in living cells and provide an accurate look at functional interactions within chromosome loci. One of the problems that plagued these functional studies was the presence of non-specific interactions, which are associations present in the data that are solely due to chromosomal proximity. In the present disclosure, these non-specific intrachromosomal interactions are captured by the methods presented herein to provide useful information for assembly.

In some embodiments, intrachromosomal interactions correlate with chromosomal connectivity. In some cases, intrachromosomal data can assist in genomic assembly. In some cases, chromatin is reconstituted in vitro. Chromatin, especially histones, the major protein component of chromatin, is the most common "C" -based technique for detecting chromatin higher-order structures and structures by sequencing: under 3C, 4C, 5C and Hi-C. This can be advantageous as it is important for fixation. Chromatin is very non-specific in terms of sequence and usually results in uniform assembly throughout the genome. In some cases, the genomes of chromatin-free species can be assembled with reconstituted chromatin, thereby extending the scope of the disclosure to all domains of the organism.

The chromatin higher-order structure capture technique is summarized in Figure 2. In short, crosslinks are created between physically close genomic regions. Cross-linking of proteins (such as histones) with DNA molecules, such as genomic DNA, within chromatin is accomplished by appropriate methods described in more detail elsewhere herein or by other methods known in the art. be able to. In some cases, two or more nucleotide sequences can be crosslinked via a protein attached to one or more nucleotide sequences. One technique is to expose chromatin to UV irradiation (Gilmour et al., Proc. Nat'l. Acad. Sci. USA, 81: 4275-4279, 1984). Cross-linking of polynucleotide segments can also be performed using other techniques, such as chemical or physical (eg, optical) cross-linking. Suitable chemical cross-linking agents include, but are not limited to, formaldehyde and psoralen (Solomon et al., Proc. NatL. Acad. Sci. USA, 82: 6470-6474, 1985, Solomon et al., Cell, 53. : 937-947, 1988). For example, cross-linking can be performed by adding 2% formaldehyde to a mixture containing DNA molecules and chromatin proteins. Other examples of agents that can be used to crosslink DNA are, but are not limited to, UV, mitomycin C, nitrogen mustard, melphalan, 1,3-butadienediepoxide, cisdiaminedichloroplatinum (II) and cyclo. There is phosphamide. Optimally, the cross-linking agent will form cross-links that bridge relatively short distances, such as about 2 Å, thereby selecting close interactions that are reversible.

In some embodiments, the DNA molecule can be immunoprecipitated before or after cross-linking. In some cases, DNA molecules can be fragmented. Fragments can specifically recognize acetylated histones, such as H3, and contact them with binding partners such as antibodies that bind to them. Examples of such antibodies include, but are not limited to, anti-acetylated histone H3, which is available from Upstate Biotechnology, Lake Placid, NY. Polynucleotides from immunoprecipitation can then be collected from immunoprecipitation. Acetylated histones can be cross-linked to adjacent polynucleotide sequences prior to fragmenting chromatin. The mixture is then processed to fractionate the polynucleotides in the mixture. Division techniques are known in the art and include, for example, shearing techniques for producing smaller genomic fragments. Fragmentation can be achieved using established methods for fragmenting chromatin, including, for example, sonication, shearing and / or the use of restriction enzymes. Restriction enzymes can have restriction sites of 1, 2, 3, 4, 5 or 6 base lengths. Examples of restriction enzymes include, but are not limited to, AatII, Acc65I, AccI, AciI, AclI, AcuI, AfeI, AflII, AflIII, AgeI, AhdI, AleI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI, AseI, AsiSI, AvaI, AvaII, AvrII, BaeGI, BaeI, BamHI, BanI, BanII, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BfaI, BfuAI, BfuCI, BglI BmgBI, BmrI, BmtI, BpmI, Bpul0I, BpuEI, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaWI, BsaXI, BscRI, BscYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI Bsp1286I, BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFI, BsrGI, BsrI, BssHII, BssKI, BssSI, BstAPI, BstBI, BstEII, BstNI, BstUI, BstUI, Bst BtgZI, BtsCI, BtsI, Cac8I, ClaI, CspCI, CviAII, CviKI-1, CviQI, DdcI, DpnI, DpnII, DraI, DraIII, DrdI, EcoI, EagleI, EarI, EcoI, Eco53kI, EcoNI, EcoO109 EcoRV, FatI, FauI, Fnu4HI, FokI, FseI, FspI, HaeII, HaeIII, HgaI, HhaI, HincII, HindIII, HinfI, HinPlI, HpaI, HpaII, HphI, Hpy166II, Hpy188I, Hpy188III, Hpy99I HpyCH4V, KasI, KpnI, MboI, MboII, MfeI, MluI, MlyI, MmeI, MnlI, MscI, MseI, MslI, MspAlI, MspI, MwoI, NaeI, NarI, Nb.BbvCI, Nb.BsmI, Nb.B. B tsI, NciI, NcoI, NdeI, NgoMIV, NheI, NlaIII, NlaIV, NmeAIII, NotI, NruI, NsiI, NspI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.C PacI, PaeR7I, PciI, PflFI, PflMI, PhoI, PleI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspGI, PspOMI, PspXI, PstI, PvuI, PvuII, RsaI, RsrII, SacI, SacII, Sal Sau96I, SbfI, ScaI, ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SspI, StuI, StyD4I, StyI, SwaI, T, TaqαI, TfiT There are Tsp45I, Tsp509I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XmaI, XmnI, and ZraI. The resulting fragments may vary in size. The resulting fragment can also contain a single chain overhang at the 5'or 3'end.

In some embodiments, sonication techniques can be used to obtain fragments of about 100-5000 nucleotides. Alternatively, fragments of about 100-1000, about 150-1000, about 150-500, about 200-500, or about 200-400 nucleotides can be obtained. Samples can be prepared for sequencing of crosslinked binding sequence segments. In some cases, a single short stretch of a polynucleotide can be created, for example by ligating two intramolecularly crosslinked sequence segments. Sequencing information can be obtained from the sample using any suitable sequencing technique described in more detail elsewhere herein, such as high throughput sequencing techniques, or another technique known in the art. Can be done. For example, the ligation product can be subjected to pair-end sequencing to obtain sequence information from each end of the fragment. Pairs of sequence segments can be represented by the resulting sequence information and correlate halotype determination information beyond the linear distance separating the two sequence segments along the polynucleotide.

One of the characteristics of the data generated by Hi-C is that when mapped to the genome, most read pairs are found to be in close linear proximity. That is, it turns out that most of the read pairs are close to each other in the genome. In the resulting dataset, the probability of intrachromosomal contact is, on average, much higher than that of interchromosomal contact, as would be expected if the chromosomes occupy a separate territory. Moreover, the probability of interaction decreases rapidly with linear distance, but even loci separated by> 200 Mb on the same chromosome are more likely to interact than loci on different chromosomes. In the detection of long-range intrachromosomal and especially interchromosomal contacts, this "background" of short- and medium-range intrachromosomal contacts is the background noise that should be subtracted using Hi-C analysis.

In particular, Hi-C experiments in eukaryotes showed two standard patterns of interaction, in addition to species-specific and cell-type-specific chromatin interactions. The first pattern, distance-dependent decline (DDD), is a common trend of decline in interaction frequency as a function of genomic distance. The second pattern, the cis-trans ratio (CTR), is markedly between loci located on the same chromosome relative to loci on different chromosomes, even when separated by sequences of tens of megabases. High interaction frequency. These patterns may reflect general polymer mechanics, where proximal loci are more likely to interact randomly and tend to occupy separate volumes within the nucleus with little mixing. It has specific nuclear organization characteristics such as the formation of chromosomal territories, which is a phenomenon of interphase chromosomes. The exact details of these two patterns can vary between species, cell types and cell states, but they are ubiquitous and prominent. These patterns are very strong and consistent and are used to determine experimental quality and are usually standardized from the data to reveal detailed interactions. However, in the methods disclosed herein, genomic assembly can utilize the three-dimensional structure of the genome. The features that impede the analysis of specific loop interactions in standard Hi-C interaction patterns, namely their ubiquity, strength and consistency, are powerful tools for estimating the genomic position of contigs. Can be used.

In a specific practice, a study of the physical distance between intrachromosomal read pairs reveals some useful features of the data regarding genomic assembly. First, short-range interactions are more common than long-range interactions (see, eg, Figure 6). That is, each read in a read pair is more likely to pair with a region that is closer than it is to a region that is far away in the actual genome. Second, there is a long tail of intermediate and long range interactions. That is, the lead pair holds information about intrachromosomal organization at kilobase (kB) or even megabase distance (Mb). For example, a read pair can provide sequence information over a range of more than about 10 kB, about 50 kB, about 100 kB, about 200 kB, about 500 kB, about 1 Mb, about 2 Mb, about 5 Mb, about 10 Mb, or about 100 Mb. These features of the data simply indicate that regions of the genome that are close together on the same chromosome are likely to be physically close together, and these regions are chemically linked to each other by the DNA backbone. The result is as expected. It was speculated that that of genome-wide chromatin interactions, such as the dataset produced by Hi-C, could provide a long range of information on sequence grouping and linear organization along the entire chromosome.

Experimental methods of Hi-C are direct and relatively low cost, but the current procedure is 10 ^{6-10 8} cells for genomic assembly and haplotyping, especially specific human patient samples. It requires a very large amount of material that cannot be obtained from. In contrast, the methods disclosed herein include methods that provide accurate and predictive results for genotype assembly, haplotype fading and metagenomics with significantly less cell-derived material. For example, about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1.0 μg, about 1.2 μg, about 1.4 μg, About 1.6 μg, about 1.8 μg, about 2.0 μg, about 2.5 μg, about 3.0 μg, about 3.5 μg, about 4.0 μg, about 4.5 μg, about 5.0 μg, about 6.0 μg, about 7.0 μg, about 8.0 μg, about 9.0 μg, about 10 μg, about 15 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 150 μg, about 200 μg, about 300 μg, about 400 μg, about 500 μg, DNA of less than about 600 μg, about 700 μg, about 800 μg, about 900 μg, or about 1000 μg can be used in the methods disclosed herein. In some examples, the DNA used in the methods disclosed herein is about 1,000,000, about 500,000, about 100,000, about 50,000, about 10,000, about 5,000, about 1,000, about 5,000, or about 1,000, about 500. , Or can be extracted from less than about 100 cells.

In general, procedures for probing the physical layout of chromosomes, such as Hi-C-based techniques, utilize chromatin formed in cells / organisms, such as chromatin isolated from cultured cells or primary tissues. The present disclosure provides for the use of such techniques in reconstituted chromatin as well as chromatin isolated from cells / organisms. Reconstituted chromatin is distinguished from chromatin formed in cells / organisms for various characteristics. First, for many samples, collecting naked DNA samples can be done in a variety of non-invasive to invasive ways, such as collecting body fluids, wiping the oral or rectal area with a cotton swab, or collecting epithelial samples. It can be realized by using it. Second, chromatin rearrangement substantially impedes the formation of interchromosomal and other long-range interactions that produce artificial products for genomic assembly and haplotype fading. In some cases, according to the methods and compositions of the present disclosure, the sample will be about 20, 15, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.4. , 0.3, 0.2, less than 0.1% or less interchromosomal or intermolecular crosslinks. In some examples, the sample can have less than about 5% interchromosomal or intermolecular crosslinks. In some examples, the sample can have less than about 3% interchromosomal or intermolecular crosslinks. In a further example, it can have less than about 1% interchromosomal or intermolecular crosslinks. Third, the frequency of sites capable of cross-linking, and thus the frequency of intramolecular cross-linking within the polynucleotide, can be adjusted. For example, the ratio of DNA to histones can be varied so that the nucleosome density can be adjusted to the desired value. In some cases, nucleosome density is reduced below physiological levels. Therefore, the distribution of crosslinks can be modified to favor longer range of interactions. In some embodiments, subsamples with varying crosslink densities can be prepared to cover both long and short range of associations. For example, the cross-linking conditions are at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%. , About 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 100% crosslinks are at least about 50 kb, about 60 kb, about 70 kb, about 70 kb on the sample DNA molecule. 80kb, about 90kb, about 100kb, about 110kb, about 120kb, about 130kb, about 140kb, about 150kb, about 160kb, about 180kb, about 200kb, about 250kb, about 300kb, about 350kb, about 400kb, about 450kb or about 500kb away It can be adjusted to occur between DNA segments that are cross-linked.

In various embodiments, the present disclosure provides various methods that allow mapping of multiple read pairs to multiple contigs. There are several open house computer programs for mapping reads to contig arrays. These read mapping program data also provide data that describes how unique a particular read mapping is within the genome. From a population of leads that are uniquely mapped within the contig with high reliability, the distribution of distances between leads can be inferred for each lead pair. These are the data shown in Figure 6. For lead pairs where the leads are clearly mapped to different contigs, this mapping data means the connection between the two contigs in question. It also means the distance between the two contigs, which is proportional to the distance distribution found from the above analysis. Therefore, each lead pair whose leads are mapped to different contigs means the connection between those two contigs in the correct assembly. The connections inferred from all such mapped read pairs can be summarized in an adjacency matrix, where each contig is represented by both rows and columns. The lead pair connecting the contigs is marked as a non-zero value in the corresponding row and column indicating the contig to which the leads are mapped in the lead pair. Most lead pairs can be mapped within the contig, from which the distribution of distances between the lead pairs can be known, from which lead pairs mapped to different contigs can be used to construct the adjacency matrix of the contig.

In various embodiments, the present disclosure provides a method comprising the step of constructing a contig adjacency matrix using read mapping data from read-to-data. In some embodiments, the adjacency matrix uses a weighting scheme for lead pairs that incorporates a tendency for short-range interactions rather than long-range interactions (see, eg, Figure 3). Lead pairs that span shorter distances are usually more common than lead pairs that span longer distances. Maps describing the probabilities of a particular distance can be matched using read-to-data mapped to a single contig to know this distribution. Therefore, one of the key features of read pairs that are mapped to different contigs is their position on the contig to which they are mapped. For lead pairs both mapped near one end of the contig, the estimated distance between these contigs can be short, and therefore the distance between the joined leads is small. Since the distance between lead pairs is generally shorter than long, this configuration provides stronger evidence that these two contigs are adjacent rather than the leads being mapped far away from the contig edge. offer. Therefore, the connections in the adjacency matrix are further weighted by the lead distance to the end of the contig. In a further embodiment, the adjacency matrix can be further rescaled to reduce the weight of many contacts on some contigs that represent indiscriminate regions of the genome. These regions of the genome, which can be identified by the high proportion of read mappings, are likely to contain false read mappings, which can speculatively give false information to the assembly. In yet other embodiments, this scaling is one or more for one or more agents that regulate the scaffold interaction of chromatin, such as transcriptional repressor CTCF, endocrine receptors, cohesins or covalently modified histones. It can be derived by searching for the conservative binding site of.

In some embodiments, the disclosure comprises one or more methods disclosed herein comprising the step of analyzing an adjacency matrix to determine its order and / or direction through a contig, indicating its direction with respect to the genome. I will provide a. In other embodiments , the route through the contig can be selected to visit ( call) each contig exactly once . In a further embodiment, the route through the contig is selected so that the route through the adjacency matrix maximizes the total number of branch weights visited ( called ) . In this way, the most reliable contig connection is proposed as the correct assembly. In yet another embodiment , each contig can be visited ( called ) exactly once and the route through the contig can be selected so that the branch weighting of the adjacency matrix is maximized.

In the diploid genome, it is often important to know which allelic variants are linked on the same chromosome. This is known as haplotype fading. Short reads of high-throughput sequence data allow rare direct observation of which allelic variant is linked. Calculating haplotype fading inferences can be unreliable over long distances. The present disclosure provides one or more methods that allow the use of allelic variants on a lead pair to determine which allelic variant is linked.

In various embodiments, the methods and compositions of the present disclosure allow haplotype fading of diploid or polyploid genomes for variants of multiple alleles. Accordingly, the methods described herein provide that determination of allelic variants that are linked is linked based on variant information of contigs assembled using lead pairs and / or the same read pair. can do. Examples of allelic variants include, but are not limited to, those known from the 1000 Genomes, UK10K, HapMap and other projects for discovering genetic variants between humans. As demonstrated, disease associations for specific genes have haplotype fading data, for example, unlinked inactivated mutations (Lupski JR, Reid JG) in both copies of SH3TC2 that result in Charcot-Marie-tooth neuropathy. , Gonzaga-Jauregui C et al., N. Engl. J. Med. 362: 1181-91, 2010) and unlinked inactivated mutations in both copies of ABCG5 resulting in hypercholesterolemia 9 (, It can be clarified more easily by the discovery of Rios J, Stein E, Shendure J et al., Hum. Mol. Genet. 19: 4313-18, 2010).

Humans are heterozygous at one in 1,000 sites on average. In some cases, single-lane data using high-throughput sequencing methods can generate at least about 150,000,000 read pairs. Read pairs can be about 100 base pairs in length. From these parameters, it is estimated that 1/10 of all reads from human samples cover heterozygous sites. Therefore, on average, 1/100 of all read pairs from human samples are estimated to cover a pair of heterozygous sites. Therefore, about 1,500,000 read pairs (1/100 of 150,000,000) using a single lane provide fading data. If there are approximately 3,000,000,000 bases in the human genome, and one in 1,000 bases is heterozygous, there are approximately 3,000,000 heterozygous sites in the average human genome. For approximately 1,500,000 read pairs representing a pair of heterozygous sites, the average coverage of each heterozygous site attempted to fade using a single lane in the high throughput sequencing method is a typical high throughput sequencer. Using, is about (1 ×). Thus, the diploid human genome can be reliably and completely faded with one-lane high-throughput sequence data associated with sequence variants from samples prepared using the methods disclosed herein. In some examples, one lane of data can be a set of DNA sequence read data. In a further example, one lane of data can be a set of DNA sequence read data from a single run of a high throughput sequencing instrument.

Since the human genome consists of two sets of homologous chromosomes, an overview of maternal and paternal copies or haplotypes of genetic material is needed to understand the true genetic structure of an individual. Obtaining an individual haplotype is useful in several ways. First, haplotypes are clinically useful in predicting donor-host adaptation outcomes in organ transplantation and are increasingly being used as a means of detecting disease associations. Second, in genes that exhibit complex heterozygotes, haplotypes provide information on whether two harmful variants are located on the same allele and predict whether the inheritance of these variants is harmful. It has a big impact. Third, haplotypes of individual groups have provided information on population structure and the evolutionary history of humankind. Finally, the recently described widespread allele imbalance in gene expression suggests that genetic or posterior differences between alleles can contribute to quantitative differences in expression. .. Understanding the haplotype structure will explain in detail the mechanism of variants that contribute to allelic imbalances.

In certain embodiments, the methods disclosed herein include in vitro techniques for fixing and capturing associations between distant regions of the genome, depending on the need for long-range ligation and fading. In some cases, the method comprises constructing and sequencing an XLRP library to supply genomically very distant read pairs. In some cases, the interaction primarily results from random association within a single DNA fragment. In some examples, segments that are close to each other within a DNA molecule interact more frequently and with a higher probability, but interactions between distant parts of the molecule are less frequent, so the genomic distance between the segments is reduced. You can guess. Therefore, there is a systematic relationship between the number of pairs connecting the two loci and their proximity on the input DNA. As demonstrated in FIG. 2, the present disclosure allows the creation of read pairs that can span the largest DNA fragment in extraction. The input DNA for this library has a maximum length of 150 kbp, which is the longest meaningful read pair observed from the sequencing data. This suggests that this method can ligate further genomically distant loci when larger input DNA fragments are obtained. Complete genomic assembly may be possible by applying improved assembly software tools specifically adapted to handle the types of data produced by this method.

The data produced using the methods and compositions of the present disclosure can achieve extremely high fading accuracy. Compared to previous methods, the methods described herein are capable of fading a higher percentage of variants. Fading can be achieved while maintaining a high level of accuracy. This phase information has a longer range, eg about 200kbp, about 300kbp, about 400kbp, about 500kbp, about 600kbp, about 700kbp, about 800kbp, about 900kbp, about 1Mbp, about 2Mbp, about 3Mbp, about 4Mbp, about 5Mbp, or It can be extended longer than about 10 Mbps. In some embodiments, more than 90% of the heterozygous SNPs in human samples use less than about 250,000,000 reads or lead pairs, eg by using data from only one lane of Illumina HiSeq 99. Fading can be performed with higher accuracy than%. In other cases, more than about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the heterozygous SNPs in human samples are about 250,000,000 or less than about 500,000,000 reads. Alternatively, lead pairs can be used, for example, by using data from only one or two lanes of Illumina HiSeq, fading with greater accuracy than about 70%, 80%, 90%, 95% or 99%. For example, more than 95% or 99% of heterozygous SNPs in human samples can be faded with greater accuracy than about 95% or 99% using less than about 250,000,000 or about 500,000,000 reads. In more cases, increase the read length to about 200bp, 250bp, 300bp, 350bp, 400bp, 450bp, 500bp, 600bp, 800bp, 1000bp, 1500bp, 2kbp, 3kbp, 4kbp, 5kbp, 10kbp, 20kbp, 50kbp or 100kbp. Allows further variants to be captured.

In other embodiments of the present disclosure, data from the XLRP library can be used to confirm the fading capability of a long range of read pairs. As shown in Figure 6, the accuracy of these results is comparable to the best technology available so far, but has been further extended to significantly longer distances. Current sample preparation procedures for specific sequencing methods recognize variants located within the read length of the restricted site targeted for fading, eg 150 bp. In one example, 44% of the 1,703,909 heterozygous SNPs present were faded with greater than 99% accuracy from the XLRP library assembled against the assembly reference sample NA12878. In some cases, this proportion can be extended to almost all variable sites by wise selection of restriction enzymes or combinations of different enzymes.

In some embodiments, the compositions and methods described herein allow for the investigation of metagenomics found, for example, in the human intestine. Therefore, partial or whole genomic sequences of some or all organisms living in a given ecological environment can be investigated. Examples include random sequencing of all intestinal microorganisms, microorganisms found in specific areas of the epidermis and microorganisms living in toxic waste sites. The composition of microbial populations in these environments can be determined using the compositions and methods described herein, as well as interrelated biochemical embodiments encoded by their respective genomes. The methods described herein are, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80, Complex organisms containing 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000 or more and / or variants of organisms. It can enable metagenomic research from a scientific environment.

The high accuracy required for sequencing cancer genomes can be achieved using the methods and systems described herein. Inaccurate reference genomes can be a challenge for base calling when sequencing the cancer genome. Non-uniform samples and small starting materials, such as samples obtained by biopsy, pose additional challenges. Moreover, the detection of large-scale structural variants and / or loss of heterozygosity is often critical for the sequencing of the cancer genome, as well as the ability to differentiate between somatic variants and errors in base calling.

The systems and methods described herein are from complex samples containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more various genomes. It is possible to generate an accurate long array. Mixed samples of normal, benign and / or tumor origin can be optionally analyzed without the need for a normal control. In some embodiments, starting samples of only 100 ng or even hundreds of genomic equivalents are utilized to generate accurate long sequences. The systems and methods described herein can enable the detection of large scale structural variants and reorganizations. Faded variant call, about 1kbp, about 2kbp, about 5kbp, about 10kbp, 20kbp, about 50kbp, about 100kbp, about 200kbp, about 500kbp, about 1Mbp, about 2Mbp, about 5Mbp, about 10Mbp, about 20Mbp, about 50Mbp or A phase variant call can be obtained for a long sequence spanning about 100 Mbps or longer nucleotides, eg, a phase variant call can be obtained for a long sequence spanning about 1 Mbps or about 2 Mbps.

Haplotypes determined using the methods and systems described herein can be allocated to computational resources, such as computational resources on networks such as cloud systems. Short variant calls can be modified as needed using the relevant information stored in the computational resources. Structural variants can be detected based on complex information from short variant calls and information stored in computational resources. Segment duplication, structurally deformable regions, highly variable and medically relevant MHC regions, centromere and telomere regions, and, but not limited to, repeat regions, low sequence accuracy, high variant rates, ALU repeats, segment duplication. Alternatively, problematic genomic moieties such as other heterologous regions, including regions with any other relevant problematic moieties known in the art, can be reassembled for increased accuracy.

Sample types can be assigned to array information in either local or networked computational resources such as the cloud. If the source of information is known, for example, if the source of information is from cancer or normal tissue, the source can be assigned to the sample as part of the sample type. Examples of other sample types usually include, but are not limited to, tissue type, sample collection method, presence of infection, type of infection, treatment method, sample size, and the like. When a complete or partial comparative genome sequence, such as a normal genome to be compared with the cancer genome, is available, the difference between the sample data and the comparative genome sequence can be determined and output at will.

The method can be used in the analysis of genetic information of a selective genomic region of interest and genomic regions that can interact with the selective region of interest. The amplification methods disclosed herein are not limited to those found in US Pat. Nos. 6,449,562, 6,287,766, 7,361,468, 7,414,117, 6,225,109 and 6,110,709. It can be used with devices, kits and methods known to the person. In some cases, the amplification methods of the present disclosure can be used to amplify the target nucleic acid for DNA hybridization studies, thereby determining the presence or absence of polymorphisms. Polymorphs or alleles can be associated with a disease or condition, such as a genetic disorder. In other cases, polymorphisms may be associated with susceptibility to the disease or condition, eg, addiction, degenerative and age-related conditions, polymorphisms associated with cancer, and the like. In other cases, polymorphism may be associated with beneficial traits such as coronary artery health promotion, or resistance to diseases such as HIV or malaria, or resistance to degenerative diseases such as osteoporosis, Alzheimer's disease or dementia. ..

The compositions and methods of the present disclosure can be used for diagnostic, prognosis, treatment, patient stratification, drug development, treatment selection and screening purposes. The present disclosure provides the advantage that many different target molecules can be analyzed at one time from a single biomolecule sample using the methods of the present disclosure. This allows, for example, several diagnostic tests to be performed on a single sample.

The compositions and methods of the present disclosure can be used for genomic studies. The methods described herein can promptly provide a highly desirable answer for this application. The methods and compositions described herein can be used in the process of finding biomarkers that can be used for diagnosis or prognosis and as indicators of health and disease. The methods and compositions described herein can be used to screen for drugs, eg, drug development, treatment selection, therapeutic efficacy determination, and / or drug development targets. Since proteins are the ultimate gene product in the body, the ability to test gene expression in drug-containing screening assays is crucial. In some embodiments, the methods and compositions described herein will measure the expression of both proteins and genes simultaneously, providing the most information in that a particular screening will be performed. Will be.

The compositions and methods of the present disclosure can be used for gene expression analysis. The methods described herein discriminate between nucleotide sequences. Differences between target nucleotide sequences can be, for example, single nucleobase differences, nucleic acid deletions, nucleic acid insertions or rearrangements. Such sequence differences containing two or more bases can also be detected. The process of the present disclosure is capable of detecting infectious diseases, genetic diseases and cancers. It is also useful in environmental monitoring, forensics and food science. Examples of gene analysis that can be performed on nucleic acids include, for example, SNP detection, STR detection, RNA expression analysis, promoter methylation, gene expression, virus detection, virus subclassification and drug resistance.

This method can be applied to the analysis of biomolecular samples obtained from or derived from patients to determine whether diseased cell types are present in the samples, stage, patient prognosis, and patient specific treatment. The ability to respond or the best treatment for the patient can be determined. The method can also be applied to identify biomarkers for specific diseases.

In some embodiments, the methods described herein are used for diagnosing a condition. As used herein, the term "diagnosing" or "diagnosing" a condition refers to predicting or diagnosing the condition, determining a predisposition to the condition, monitoring the treatment of the condition, and the therapeutic response of the disease. It can include diagnosing, or the prognosis of the condition, the progression of the condition, or the response to a particular treatment of the condition. For example, blood samples are assayed according to any of the methods described herein to determine the presence and / or amount of disease markers or malignant cell types in the sample, thereby diagnosing or grading the disease or cancer. can do.

In some embodiments, the methods and compositions described herein are used for diagnosis and prognosis of the condition.

A large number of immune, proliferative, malignant diseases and disorders are particularly suitable for the methods described herein. Immune diseases and disorders include allergic diseases and disorders, disorders of immune function and autoimmune diseases and conditions. Allergic diseases and disorders include, but are not limited to, allergic rhinitis, allergic conjunctivitis, allergic asthma, atopic eczema, atopic dermatitis and dietary allergies. Immunodeficiency includes, but is not limited to, severe combined immunodeficiency (SCID), neutropenia syndrome, chronic granulomatous disease, leukocyte adhesion deficiency I and II, high IgE syndrome, Chediak-Higashi syndrome, There are neutrophil hyperplasia, neutropenia, dysplasia, agammaglobulinemia, hyper-IgM syndrome, DiGeorge / palatal cardiac facial syndrome and interferon γTH1 pathway deficiency. Autoimmune and immune dysregulation disorders include, but are not limited to, rheumatoid arthritis, diabetes, systemic erythematosus, Graves' disease, Grave's eye disorder, Crohn's disease, multiple sclerosis, psoriasis, systemic sclerosis, thyroid tumor. And lymphomatosis thyroidoma (Hashimoto's disease, lymphoma thyroidoma), alopecia round, autoimmune myocardial disease, sclerosing lichen, autoimmune vasculitis, Azison's disease, atrophic gastric inflammation, severe myasthenia, Idiopathic thrombocytopenic purpura, hemolytic anemia, primary biliary cirrhosis, Wegener's granulomatosis, nodular polyarteritis and inflammatory bowel disease, allogeneic transplant rejection and allergic reactions to infectious microorganisms or environmental antigens There is tissue destruction due to.

Proliferative disorders and disorders that can be assessed by the methods of the present disclosure include, but are not limited to, hemangiomasosis, secondary advanced multiple sclerosis, chronic progressive myelopathy, neurofibrosis, ganglia in neonates. There are swelling, keloid formation, bone Paget's disease, fibrous cystic disease (eg, breast or uterine), sarcoidosis, pelony and dupuytran fibrosis, cirrhosis, atherosclerosis and vascular restenosis.

Malignant diseases and disorders that can be evaluated by the methods of the present disclosure include hematological malignancies and solid tumors.

Since such malignancies contain changes in blood-derived cells, hematological malignancies are particularly suitable for the methods of the present disclosure if the sample is a blood sample. Such malignant tumors include non-Hodgkin lymphoma, Hodgkin lymphoma, non-B cell line lymphoma and other lymphomas, acute or chronic leukemia, erythrocytosis, thromboemia, multiple myeloma, myeloma dysplasia, myeloproliferative disorders. There are sexual disorders, myeloma fibrosis, atypical immune lymphocyte proliferation and plasma cell disorders.

Plasma cell disorders that can be assessed by the methods of the present disclosure include multiple myeloma, amyloidosis and Waldenström macroglobulinemia.

Examples of solid tumors include, but are not limited to, colon cancer, breast cancer, lung cancer, prostate cancer, brain tumor, central nervous system tumor, bladder tumor, melanoma, liver cancer, osteosarcoma and other bone cancers, testicles and ovaries. There are cancers, head and neck tumors and cervical neoplasms.

Genetic disorders can also be detected by the process of the present disclosure. This can be done by prenatal or postnatal screening for chromosomal and genetic abnormalities or diseases. Examples of detectable genetic disorders include 21-hydroxylase deficiency, cystic fibrosis, fragile X syndrome, Turner syndrome, Duchenne muscular dystrophy, Down syndrome or other trisomy, heart disease, monogenic disease, HLA. There are typing, phenylketonuria, sickle erythrocyte anemia, Teisax's disease, salacemia, Kleinfelder's syndrome, Huntington's disease, autoimmune disease, lipidosis, obesity, hemophilia, congenital metabolic disorders and diabetes.

The methods described herein are used to diagnose pathogenic infections, such as intracellular bacterial and viral infections, by determining the presence and / or amount of markers for each of the bacteria or viruses in the sample. be able to.

A wide variety of infectious diseases can be detected by the process of the present disclosure. Infectious diseases can be caused by infectious agents of bacteria, viruses, parasites and fungi. The resistance of various infectious agents to the drug can also be determined using this disclosure.

Bacterial infectious agents that can be detected by the present disclosure include Escherichia coli, Salmonella, Shigella, KlESBiella, Pseudomonas, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium intracellulare, Yersinia, Francisella, Pasteurella, Brucella, Clostridia, Pertussis Bacteria pertussis, Bacteroides, Staphylococcus aureus, Streptococcus pneumonia, Group B hemolytic streptococcus (B-Hemolytic strep.), Corynebacteria, Legionella ), Mycoplasma, Ureaplasma, Chlamydia, Neisseria gonorrhea, Neisseria meningitides, Hemophilus influenza, Enterococcus faecalis, Proteus Proteus vulgaris, Proteus mirabilis, Helicobacter pylori, Treponema bacteria, Borrelia burgdorferi, Borrelia recurrentis, Borrelia recurrentis There are pathogens, Nocardia and Actinomycetes.

Fungal infectious agents that can be detected by the present disclosure include Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum, and Coccidiides immitis. , Paracoccidiides brasiliensis, Candida albicans, Aspergillus fumigautus, Algae [Rhizopus] (Phycomycetes) There are Chromomycosis and Maduromycosis.

Viral infectious agents that can be detected by the present disclosure include human immunodeficiency virus, human T-cell lymphophilic virus, hepatitis virus (eg, hepatitis B virus and hepatitis C virus), Epsteiner virus, cytomegalo. There are viruses, human papillomavirus, orthomixovirus, paramixovirus, adenovirus, coronavirus, rabdovirus, poliovirus, togavirus, bunyavirus, arenavirus, eczema virus and leovirus.

Parasitic factors that can be detected by the present disclosure include Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, and Plasmodium ovale. ), Onchoverva volvulus, Leishmania, Tripanosoma, Livestock sucker, Entamoeba histolytica, Cryptospolidium, Diargia, Tricomonas, Balatidium coli, Wuchereria bancrofti ), Toxoplasma, Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculus medinesis, Sucker, Diphyllobothrium latum, Plasmodium, Plasmodium There are Carinii (Pneumocystis carinii) and American Plasmodium (Necator americanis).

The present disclosure is also useful for detecting drug resistance by infectious agents. For example, all of the bancomycin-resistant Enterococcus faecium, methicillin-resistant yellow bacillus, penicillin-resistant Streptococcus pneumoniae, multidrug-resistant human tuberculosis, and AZT-resistant human immunodeficiency virus. It can be identified in the disclosure.

Therefore, the target molecule detected using the compositions and methods of the present disclosure can be either a patient marker (such as a cancer marker) or an infectious disease marker due to a foreign factor such as a bacterial or viral marker.

The compositions and methods of the present disclosure are used to identify and / or quantify target molecules whose abundance is an indicator of biological status or pathology, such as blood markers that are upregulated or downregulated as a result of the pathology. be able to.

In some embodiments, the methods and compositions of the present disclosure can be used for cytokine expression. The low sensitivity of the methods described herein can be useful, for example, for early detection of cytokines as biomarkers for the diagnosis or prognosis of diseases such as cancer, and identification of potential states.

Different samples from which the target polynucleotide is obtained can include multiple samples from the same individual, samples from different individuals or combinations thereof. In some embodiments, the sample comprises multiple polynucleotides of one individual origin. In some embodiments, the sample comprises a plurality of polynucleotides from two or more individuals. An individual is any organism or part thereof from which a target polynucleotide can be obtained, and examples thereof include, but are not limited to, plants, animals, fungi, prokaryotes, prokaryotes, viruses, mitochondria, and chloroplasts. Sample polynucleotides can be isolated from a subject, such as a cell sample, tissue sample or organ sample obtained from the subject, including, for example, cultured cell lines, biopsies, blood samples or liquid samples containing cells. The subject is an animal, which may include, but is not limited to, animals such as cows, pigs, mice, rats, chickens, cats, dogs and the like, and is usually mammals such as humans. Samples can also be obtained artificially, such as by chemical synthesis. In some embodiments, the sample comprises DNA. In some embodiments, the sample comprises genomic DNA. In some embodiments, the sample comprises mitochondrial DNA, chlorophyll DNA, plasmid DNA, bacterial artificial chromosome, yeast artificial chromosome, oligonucleotide tag or a combination thereof. In some embodiments, the sample is DNA produced by a primer extension reaction using any suitable combination of primers and DNA polymerase, including but not limited to polymerase chain reaction (PCR), reverse transcription and combinations thereof. including. When the template for the primer extension reaction is RNA, the reverse transcript is called complementary DNA (cDNA). Primers useful for the primer extension reaction can include one or more target-specific sequences, random sequences, partially random sequences and combinations thereof. Reaction conditions suitable for the primer extension reaction are known in the art. In general, the sample polynucleotide includes any polynucleotide present in the sample and may or may not include the target polynucleotide.

In some embodiments, the template nucleic acid molecule (eg, DNA or RNA) is isolated from a biological sample containing various other components such as proteins, lipids and non-template nucleic acids. The template nucleic acid molecule can be obtained from any cellular material and can be obtained from animals, plants, bacteria, fungi or any other cellular organism. Biological samples used in the present disclosure include viral particles or preparations. The template nucleic acid molecule can be obtained directly from the organism or a biological sample obtained from the organism, such as blood, urine, cerebrospinal fluid, semen, saliva, sputum, stool and tissue. Any tissue or body fluid sample can be used as a source of nucleic acids used in the present disclosure. The template nucleic acid molecule can also be isolated from cultured cells such as primary cell cultures or cell lines. The cells or tissues from which the template nucleic acid is obtained can also be infected with a virus or other intracellular pathogen. The sample can also be total RNA, cDNA library, virus or genomic DNA extracted from a biological sample. The sample can also be isolated DNA of non-cellular origin, eg DNA amplified / isolated from a freezer.

Nucleic acid extraction and purification methods are well known in the art. For example, nucleic acids can be purified by organic extraction with phenol, phenol / chloroform / isoamyl alcohol, or similar formulations containing TRIzol and TriReagent. Other unrestricted examples of extraction techniques include: (1) with or without an automated nucleic acid extractor, eg, Model 341 DNA Extractor available from Applied Biosystems (Foster City, Calif.). For example, using phenol / chloroform organic reagents (Ausubel et al., 1993), organic extraction with ethanol precipitation, (2) stationary phase adsorption method (US Pat. No. 5,234,809, Walsh et al., 1991), and (3) salts. Inducible Nucleic Acid Precipitation Method (Miller et al. (1988), such precipitation method is commonly referred to as the "salting" method. Another example of isolating and / or purifying nucleic acid is the use of magnetic particles. , Nucleic acid can be specifically or non-specifically bound to its particles, after which the beads can be isolated and washed using a magnet and the nucleic acid can be eluted from the beads (see, eg, US Pat. No. 5,705,628). ). In some embodiments, there may be the above isolation method after an enzymatic digestion step, eg digestion with Proteinase K or other protease, which helps remove unwanted proteins from the sample, eg US Pat. No. 7,001,724. See No. If desired, an RNase inhibitor can be added to the lysis buffer. For certain cell or sample types, it is desirable to add a protein denaturation / digestion step to the procedure. The method can cover the isolation of DNA, RNA or both. If both DNA and RNA are isolated together during or after the extraction procedure, a further step is utilized to take advantage of one or both. Can be purified separately from others. For example, purification by size, sequence or other physical or chemical properties can also produce a fraction of the extracted nucleic acid. In the first nucleic acid isolation step. In addition, after any step in the methods of the present disclosure, nucleic acid purification can be performed, such as removing excess or unwanted reagents, reactants or products.

Template nucleic acid molecules can be obtained as described in US Patent Application Publication No. 2002 / 0,190,663 A1 published October 9, 2003. Nucleic acids can usually be extracted from biological samples by a variety of techniques, including those described in Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, N.Y., pp. 280-281 (1982). In some cases, the nucleic acid is the first extract from a biological sample and then can be crosslinked in vitro. In some cases, naturally associated proteins (eg, histones) can be further removed from the nucleic acid.

In other embodiments, the present disclosure facilitates any high molecular weight double-stranded DNA, including DNA isolated from, for example, tissues, cell cultures, body fluids, animal tissues, plants, bacteria, fungi, viruses and the like. Can be applied.

In some embodiments, each of the plurality of independent samples is at least about 1 ng, 2 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 75 ng, 100 ng, 150 ng, 200 ng, 250 ng, 300 ng, 400 ng, 500 ng. , 1 μg, 1.5 μg, 2 μg, 5 μg, 10 μg, 20 μg, 50 μg, 100 μg, 200 μg, 500 μg, or 1000 μg, or more nucleic acid materials, respectively, can be independently included. In some embodiments, each of the plurality of independent samples is approximately 1 ng, 2 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 75 ng, 100 ng, 150 ng, 200 ng, 250 ng, 300 ng, 400 ng, 500 ng, It can independently contain less than or less than 1 μg, 1.5 μg, 2 μg, 5 μg, 10 μg, 20 μg, 50 μg, 100 μg, 200 μg, 500 μg, or 1000 μg, respectively.

In some embodiments, end repairs are performed using commercially available kits, such as those available from Epicentre Biotechnologies (Madison, WI), to produce blunt-ended 5'phosphorylated nucleic acid ends.

Adapter oligonucleotides include any oligonucleotide that is at least partially known and has a sequence capable of conjugating to the target polynucleotide. Adapter oligonucleotides can include DNA, RNA, nucleotide analogs, non-standard nucleotides, labeled nucleotides, modified nucleotides or combinations thereof. The adapter oligonucleotide can be single-stranded, double-stranded or partially double-stranded. In general, a partial double-stranded adapter comprises one or more single-stranded regions and one or more double-stranded regions. Double-stranded adapters can contain two separate oligonucleotides that hybridize to each other (also referred to as "oligonucleotide double strands"), and by hybridization, one or more blunt ends, one or more 3'. Overhangs, one or more 5'protrusions, mismatches and / or one or more bulges due to unpaired nucleotides, or any combination thereof, may remain. In some embodiments, the single-stranded adapter comprises two or more sequences that can hybridize to each other. Hybridization results in a hairpin structure when two such hybridizable sequences are contained in a single-stranded adapter (hairpin adapter). When the two hybridized regions of the adapter are separated from each other by non-hybridized regions, a "bubble" structure occurs. Adapters containing bubble structures can consist of a single adapter oligonucleotide containing internal hybridization, or can include two or more adapter oligonucleotides that hybridize to each other. Hybridization of internal sequences, such as between two hybridizable sequences in an adapter, can create a double-stranded structure on a single-stranded adapter oligonucleotide. Different types of adapters can be used in combination, such as hairpin adapters and double-stranded adapters, or adapters with different arrangements. The hybridizable sequence in the hairpin adapter may or may not include one or both ends of the oligonucleotide. If neither end is included in the hybridizable sequence, both ends are "free" or "protruding". If only one end is capable of hybridizing to another sequence in the adapter, the other end forms a protrusion, such as a 3'protrusion or a 5'protrusion. If both the 5'end nucleotide and the 3'end nucleotide are included in a hybridizable sequence, such that the 5'end nucleotide and the 3'end nucleotide are complementary and hybridize with each other, the end is said to be "smooth". Called. Different adapters can be conjugated to the target polynucleotide either sequentially or simultaneously. For example, first and second adapters can be added to the same reaction. The adapter can be manipulated prior to combining with the target polynucleotide. For example, terminal phosphoric acid can be added or removed.

Adapters are among one or more amplification primer annealing sequences or complements thereof, one or more sequencing primer annealing sequences or complements thereof, one or more barcode sequences, multiple different adapters or subsets of different adapters. One or more common sequences shared, one or more restriction enzyme recognition sites, one or more protrusions complementary to one or more target polynucleotide overhangs, one or more probe binding sites (eg, Illumina Inc) Tableed in a pool of adapters containing one or more random or nearly random sequences (eg, random sequences), for attachment to sequencing platforms such as flow cells for large-scale parallel sequencing, such as those developed by. Containing one or more nucleotides randomly selected from a set of two or more different nucleotides at one or more positions, each having a different nucleotide selected at one or more positions), and combinations thereof. Can contain one or more different sequence elements, but not limited to that. Two or more sequence elements can be non-adjacent to each other (eg, separated by one or more nucleotides), adjacent to each other, partially overlapping, or completely overlapping. .. For example, the amplified primer annealing sequence can also serve as a sequencing primer annealing sequence. Sequence elements can be located at or near the 3'end of the adapter oligonucleotide, near or near the 5'end, or inside. Where the adapter oligonucleotide is capable of forming a secondary structure, such as a hairpin, the sequence element is partially or completely outside the secondary structure, partially or completely inside the secondary structure, or involved in the secondary structure. Can be located between sequences that do. For example, if the adapter oligonucleotide contains a hairpin structure, the sequence element is located inside or outside the hybridizable sequence (“stem”), including within the sequence between the hybridizable sequences (“loop”). Can be targeted or perfectly located. In some embodiments, the first adapter oligonucleotide among the plurality of first adapter oligonucleotides having different barcode sequences comprises a plurality of sequence elements common among all of the first adapter oligonucleotides. In some embodiments, all of the second adapter oligonucleotides are different from the common sequence elements shared by the first adapter oligonucleotide, and are common sequence elements among all of the second adapter oligonucleotides. including. Differences in sequence elements are at least different adapters due to, for example, changes in sequence length, deletion or insertion of one or more nucleotides, or changes in nucleotide composition at one or more nucleotide positions (such as base changes or base modifications). It can be anything that some are not perfectly aligned. In some embodiments, the adapter oligonucleotide comprises a 5'protrusion, a 3'protrusion, or both that are complementary to one or more target polynucleotides. Complementary protrusions include, but are not limited to, nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or longer nucleotides, 1 It can be the above nucleotide length. For example, the complementary overhang can be about 1, 2, 3, 4, 5 or 6 nucleotides in length. Complementary protrusions can include fixed sequences. One or more represented by a pool of adapters with complementary overhangs containing random sequences so that one or more nucleotides are randomly selected from a set of two or more different nucleotides at one or more positions. Complementary protrusions with each of the different nucleotides selected at position can contain a random sequence of one or more nucleotides. In some embodiments, the adapter protrusion is complementary to the target polynucleotide protrusion produced by restriction endonuclease digestion. In some embodiments, the adapter protrusion is composed of adenine or thymine.

Adapter oligonucleotides can have at least any suitable length sufficient to contain one or more sequence elements from which they are composed. In some embodiments, the adapter is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200 or more. Less than or longer nucleotides. In some examples, the adapter can be about 10 to about 50 nucleotides in length. In a further example, the adapter can be about 20-40 nucleotides in length.

As used herein, the term "barcode" refers to a known nucleic acid sequence that makes it possible to identify some features of a polynucleotide associated with a barcode. In some embodiments, the characteristic of the polynucleotide identified is the sample from which the polynucleotide is derived. In some embodiments, the barcode can be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or longer nucleotides. For example, the barcode can be at least 10, 11, 12, 13, 14 or 15 nucleotides in length. In some embodiments, the barcode can be shorter than 10, 9, 8, 7, 6, 5 or 4 nucleotides in length. For example, barcodes can be shorter than 10 nucleotides in length. In some embodiments, the barcode associated with some polynucleotides is of a different length than the barcode associated with other polynucleotides. In general, the barcodes are of sufficient length and contain sequences that are sufficiently different to allow their identification based on the barcode to which the sample is associated. In some embodiments, the barcode and the sample source to which it is associated are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide mutations, It can be accurately identified after a mutation, insertion or deletion of one or more nucleotides in a barcode sequence, such as an insertion or deletion. In some examples, one, two or three nucleotides can be mutated, inserted and / or deleted. In some embodiments, each barcode in the plurality of barcodes has a plurality of at least two nucleotide positions, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more positions. It is different from any other barcode. In some examples, each barcode may differ from any other barcode in at least 2, 3, 4 or 5 positions. In some embodiments, both the first and second sites contain at least one of the plurality of barcode sequences. In some embodiments, the barcode of the second site is independently selected from the barcode of the first adapter oligonucleotide. In some embodiments, the first and second sites with barcodes are paired so that the pair of sequences contains one or more barcodes that are the same or different. In some embodiments, the methods of the present disclosure further comprise the step of identifying the sample from which the target polynucleotide is derived based on the barcode sequence to which the target polynucleotide is attached. In general, the barcode can include a nucleic acid sequence that, when attached to the target polynucleotide, acts as an identifier for the sample from which the target polynucleotide was derived.

In eukaryotes, genomic DNA is bundled with chromatin and organized as chromosomes in the nucleus. The basic structural unit of chromatin is the nucleosome, which is composed of 146 base pairs (bp) of DNA wrapped around a histone octamer. The histone octamer consists of two copies of each of the core histone H2A-H2B dimer and the H3-H4 dimer. Nucleosomes are regularly spaced along the DNA and are commonly referred to as "beads".

Assembly of core histones and DNA into nucleosomes is mediated by chaperone proteins and related assembly factors. Almost all of these factors are core histone binding proteins. Some histone chaperones, such as the nucleosome assembly protein-1 (NAP-1), show a preferred tendency for binding to histones H3 and H4. It was also observed that the newly synthesized histones were acetylated and subsequently assembled into chromatin and then deacetylated. Therefore, factors that mediate histone acetylation or deacetylation play an important role in the chromatin assembly process.

In general, two in vitro methods have been developed to reconstitute or assemble chromatin. One method is ATP-independent, while the second is ATP-dependent. ATP-independent methods of reconstitution of chromatin include either DNA and proteins such as core histones, plus NAP-1, or salts to act as histone chaperones. This method results in random histone organization on DNA that does not exactly mimic the natural core nucleosome particles in the cell. These particles are often referred to as mononucleosomes because they are not regularly ordered and the extended nucleosome array and the DNA sequence used usually do not exceed 250 bp (Kundu, TK et al., Mol. Cell 6). : 551-561, 2000). Chromatin must be assembled in an ATP-dependent process to generate an ordered extended array of nucleosomes on longer DNA sequences.

The ATP-dependent assembly of periodic nucleosome arrays is similar to that found in native chromatin and requires DNA sequences, core histone particles, chaperone proteins and ATP-utilized chromatin assembly factors. ACF (ATP-based chromatin assembly and remodeling factor) or RSF (remodeling and spacing factor) are two widely studied assembly factors that are used to expand and order nucleosomes in vitro. Chromatin is produced from the array of (Fyodorov, DV and Kadonaga, JT, Method Enzymol. 371: 499-515, 2003, Kundu, TK et al., Mol. Cell 6: 551-561, 2000).

In certain embodiments, the methods of the present disclosure are, for example, free DNA isolated from plasma, serum and / or urine, apoptotic DNA from cells and / or tissues, and enzymatically fragmented DNA in vitro (eg,). , DNase I and / or by restriction enzymes), and / or any type of fragmented DNA that includes, but is not limited to, DNA fragmented by mechanical force (water shearing, ultrasonic treatment, atomization, etc.). It can be easily applied to strand DNA.

Nucleic acid obtained from a biological sample can be fragmented to prepare a fragment suitable for analysis. The template nucleic acid can be fragmented or sheared to the desired length using a variety of mechanical, chemical and / or enzymatic methods. DNA can be randomly sheared by sonication, eg, Covaris method, short exposure to DNase, or a mixture of one or more restriction enzymes, or a transportase or nicking enzyme. RNA can be fragmented by short exposure to RNase, heat + magnesium or shear. RNA can be converted to cDNA. When using fragmentation, RNA can be converted to cDNA before or after fragmentation. In some embodiments, the nucleic acid derived from the biological sample is fragmented by sonication. In other embodiments, the nucleic acid is fragmented by a water shear device. Generally, individual nucleic acid template molecules can be about 2 kb to about 40 kb bases. In various embodiments, the nucleic acid can be a fragment of about 6 kb to 10 kb. The nucleic acid molecule can be single-stranded, double-stranded or double-stranded with a single-stranded region (eg, a stem-and-loop structure).

In some embodiments, the crosslinked DNA molecule can be subjected to a size selection step. Nucleic acid size selection can be performed on crosslinked DNA molecules that are smaller or larger than a particular size. Size selection can be further influenced by the frequency of cross-linking and / or the method of fragmentation, such as choosing frequent or rare cutter restriction enzymes. In some embodiments, the composition is about 1 kb to 5 Mb, about 5 kb to 5 Mb, about 5 kb to 2 Mb, about 10 kb to 2 Mb, about 10 kb to 1 Mb, about 20 kb to 1 Mb, about 20 kb to 500 kb, about 50 kb to 500 kb. , About 50 kb to 200 kb, about 60 kb to 200 kb, about 60 kb to 150 kb, about 80 kb to 150 kb, about 80 kb to 120 kb or about 100 kb to 120 kb, or any range surrounded by any of these values (for example, about 150 kb). It can be prepared to include cross-linking a DNA molecule to ~ 1Mb).

In some embodiments, the sample polynucleotide is fragmented into a population of fragmented DNA molecules in one or more specific size ranges. In some embodiments, the fragments are at least about 1, about 2, about 5, about 10, about 20, about 50, about 100, about 200, about 500, about 1000, about 2000, about 5000, about 10,000, It can be generated from about 20,000, about 50,000, about 100,000, about 200,000, about 500,000, about 1,000,000, about 2,000,000, about 5,000,000, about 10,000,000 or more genomic equivalents of starting DNA. Fragmentation can be achieved by methods known in the art, including chemical, enzymatic and mechanical fragmentation. In some embodiments, the fragments are about 10 to about 10,000, about 20,000, about 30,000, about 40,000, about 50,000, about 60,000, about 70,000, about 80,000, about 90,000, about 100,000, about 150,000, about 200,000, about. It has an average length of 300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 800,000, about 900,000, about 1,000,000, about 2,000,000, about 5,000,000, about 10,000,000 or longer nucleotides. In some embodiments, the fragments have an average length of about 1 kb to about 10 Mb. In some embodiments, the fragments are about 1 kb to 5 Mb, about 5 kb to 5 Mb, about 5 kb to 2 Mb, about 10 kb to 2 Mb, about 10 kb to 1 Mb, about 20 kb to 1 Mb, about 20 kb to 500 kb, about 50 kb to 500 kb, about 50 kb to 500 kb. 50 kb to 200 kb, about 60 kb to 200 kb, about 60 kb to 150 kb, about 80 kb to 150 kb, about 80 kb to 120 kb or about 100 kb to 120 kb, or any range surrounded by any of these values (for example, about 60 to 120 kb). Has an average length. In some embodiments, the fragment has an average length of less than about 10 Mb, less than about 5 Mb, less than about 1 Mb, less than about 500 kb, less than about 200 kb, less than about 100 kb, or less than about 50 kb. In other embodiments, the fragment has an average length of about 5 kb or more, about 10 kb or more, about 50 kb or more, about 100 kb or more, about 200 kb or more, about 500 kb or more, about 1 Mb or more, about 5 Mb or more, or about 10 Mb or more. In some embodiments, fragmentation is achieved mechanically, including subjecting the sample DNA molecule to sonication. In some embodiments, fragmentation comprises treating the sample DNA molecule with one or more enzymes under conditions suitable for one or more enzymes to produce double-stranded nucleic acid fractures. Examples of enzymes useful for the generation of DNA fragments are sequence-specific and sequence-non-specific nucleases. Non-limiting examples of nucleases include DNase I, fragmentation enzymes, restriction endonucleases, variants thereof and combinations thereof. For example, digestion with DNase I can induce random double-strand breaks in DNA in the absence of Mg ⁺⁺ and in the presence of Mn ⁺⁺ . In some embodiments, fragmentation involves treating the sample DNA molecule with one or more restriction endonucleases. Fragmentation can produce fragments with 5'protrusions, 3'protrusions, blunt ends, or combinations thereof. In some embodiments, cleavage of the sample DNA molecule leaves a protrusion with a predictable sequence, such as when fragmentation involves the use of one or more restriction endonucleases. In some embodiments, the method comprises sizing the fragment by standard methods such as column purification or isolation from an agarose gel.

In some embodiments, the 5'and / or 3'terminal nucleotide sequences of the fragmented DNA are unmodified prior to ligation. For example, fragmentation with a restriction endonuclease can be used to leave a predictable overhang and then ligate with a nucleic acid terminal containing a predictable overhang and a complementary overhang on the DNA fragment. In another example, cleavage by an enzyme that leaves a predictable blunt end can be followed by ligation of the blunt-ended DNA fragment to a nucleic acid containing the blunt end, such as an adapter, oligonucleotide or polynucleotide. In some embodiments, the fragmented DNA molecule is scraped (or "end-repaired") to a blunt end to create a DNA fragment with a blunt end and then attached to the adapter. The step of scraping the blunt ends can be accomplished by incubation with a suitable enzyme such as a DNA polymerase having both 3'→ 5'exonuclease activity and 5'→ 3'polymerase activity, such as T4 polymerase. In some embodiments, after terminal repair, one or more adenines, one or more thymines, one or more guanines or one or more cytosines, etc. 1, 2, 3, 4, 5, 6, 7, Protrusions can be created by continuing the addition of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. For example, the terminal pair can be continuously added with 1, 2, 3, 4, 5 or 6 nucleotides. In ligation reactions and the like, the overhanging DNA fragment can be conjugated to one or more nucleic acids such as oligonucleotides, adapter oligonucleotides or polynucleotides with complementary overhangs. For example, a template-independent polymerase can be used to add a single adenine to the 3'end of a terminal-repaired DNA fragment and then ligate to one or more adapters each having thymine at the 3'end. In some embodiments, nucleic acids such as oligonucleotides or polynucleotides are blunt-ended double-stranded DNAs that are modified by extending the 3'end with one or more nucleotides followed by 5'phosphorylation. Can be attached to molecules. In some cases, 3'terminal extension can be Klenow polymerase or any suitable polymerase provided herein in the presence of one or more dNTPs in a suitable buffer that can contain magnesium. It can be carried out by a polymerase such as, or by the use of terminal deoxynucleotide transferase. In some embodiments, the target polynucleotide having a blunt end is attached to one or more adapters containing the blunt end. Phosphorylation of the 5'end of the DNA fragment molecule can be performed, for example, by T4 polynucleotide kinase in a suitable buffer containing ATP and magnesium. Optionally, by using an enzyme known in the art, such as, for example, phosphatase, fragmented DNA molecules can be treated to dephosphorylate the 5'end or 3'end.

With respect to two polynucleotides, such as adapter oligonucleotides and target polynucleotides, the terms "connecting", "binding" and "ligation" herein covalently bind two separate DNA segments into a contiguous skeleton. Refers to making a single longer polynucleotide with. Methods of joining two DNA segments are known in the art and include, but are not limited to, enzymatic and non-enzymatic (eg, chemical) methods. Examples of non-enzymatic ligation reactions include the non-enzymatic ligation techniques described in US Pat. Nos. 5,780,613 and 5,476,930, which are incorporated herein by reference. In some embodiments, the adapter oligonucleotide is ligated to the target polynucleotide by a ligase, such as DNA ligase or RNA ligase. Multiple ligases, each with characterized reaction conditions, are known in the art, but not limited to tRNA ligases, Taq DNA ligases, Thermus filiformis DNA ligases, Escherichia coli DNA ligases, By Tth DNA ligase, Thermos scotoductus DNA ligase (I and II), heat resistant ligase, Ampligase heat resistant DNA ligase, VanC-type ligase, 9 ° N DNA ligase, Tsp DNA ligase and bioresource survey NAD ⁺ dependent ligase, T4 RNA ligase, T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, Pfu DNA ligase, DNA ligase 1, DNA ligase III, DNA ligase IV and biological resource survey, including novel ligases found There are ATP-dependent ligases, including novel ligases discovered by, as well as their wild forms, variant isoforms and genetically engineered variants.

Ligation is possible between DNA segments that have hybridizable sequences such as complementary overhangs. Ligation is also possible between the two blunt ends. Usually, 5'phosphoric acid is used for the ligation reaction. 5'Phosphate can be provided by the target polynucleotide, adapter oligonucleotide or both. 5'Phosphoric acid can be added to or removed from the DNA segment to be joined as needed. Methods for adding or removing 5'phosphoric acid are known in the art and include, but are not limited to, enzymatic and chemical processes. Enzymes useful for the addition and / or removal of 5'phosphate include kinases, phosphatases and polymerases. In some embodiments, both of the two ends (eg, the adapter end and the target polynucleotide end) that are joined in the ligation reaction are 5'so that two covalent bonds are created when the two ends are joined. Provides phosphoric acid. In some embodiments, only one of the two ends joined in the ligation reaction (eg, one of the adapter end and one of the target polynucleotide ends) so that only one covalent bond is created when joining the two ends. ) Provides 5'phosphoric acid.

In some embodiments, only one strand is attached to the adapter oligonucleotide at one or both ends of the target polynucleotide. In some embodiments, both strands are attached to the adapter oligonucleotide at one or both ends of the target polynucleotide. In some embodiments, 3'phosphate is removed prior to ligation. In some embodiments, adapter oligonucleotides are added to both ends of the target polynucleotide and one or both strands are attached to one or more adapter oligonucleotides at each end. If both strands are attached to the adapter oligonucleotide at both ends, the cleavage reaction can be continued after the junction, leaving a 5'protrusion that can serve as a template for extending the corresponding 3'end. , Its 3'end can or cannot contain one or more nucleotides derived from the adapter oligonucleotide. In some embodiments, the target polynucleotide is conjugated to a first adapter oligonucleotide at one end and a second adapter oligonucleotide at the other end. In some embodiments, the two ends of the target polynucleotide are attached to the opposite ends of a single adapter oligonucleotide. In some embodiments, the targeted polynucleotide and adapter oligonucleotide to be conjugated comprise a blunt end. In some embodiments, different first adapter oligonucleotides containing at least one barcode sequence are used for each sample so that no barcode sequence is attached to the target polynucleotide of more than one sample. Each sample can be subjected to a separate ligation reaction. A DNA segment or target polynucleotide having a conjugated adapter oligonucleotide is considered "tagged" by the conjugated adapter.

In some cases, the ligation reaction is about 0.1 ng / μL, about 0.2 ng / μL, about 0.3 ng / μL, about 0.4 ng / μL, about 0.5 ng / μL, about 0.6 ng / μL, about 0.7 ng / μL, about 0.8ng / μL, about 0.9ng / μL, about 1.0ng / μL, about 1.2ng / μL, about 1.4ng / μL, about 1.6ng / μL, about 1.8ng / μL, about 2.0ng / μL, Approximately 2.5 ng / μL, Approximately 3.0 ng / μL, Approximately 3.5 ng / μL, Approximately 4.0 ng / μL, Approximately 4.5 ng / μL, Approximately 5.0 ng / μL, Approximately 6.0 ng / μL, Approximately 7.0 ng / μL, Approximately 8.0 ng / μL, about 9.0 ng / μL, about 10 ng / μL, about 15 ng / μL, about 20 ng / μL, about 30 ng / μL, about 40 ng / μL, about 50 ng / μL, about 60 ng / μL, about 70 ng / μL, Approximately 80 ng / μL, Approximately 90 ng / μL, Approximately 100 ng / μL, Approximately 150 ng / μL, Approximately 200 ng / μL, Approximately 300 ng / μL, Approximately 400 ng / μL, Approximately 500 ng / μL, Approximately 600 ng / μL, Approximately 800 ng / μL or It can be carried out at a DNA segment or target polynucleotide concentration of about 1000 ng / μL. For example, ligation can be performed at DNA segment or target polynucleotide concentrations of about 100 ng / μL, about 150 ng / μL, about 200 ng / μL, about 300 ng / μL, about 400 ng / μL or about 500 ng / μL.

In some cases, the ligation reaction is about 0.1-1000 ng / μL, about 1-1000 ng / μL, about 1-800 ng / μL, about 10-800 ng / μL, about 10-600 ng / μL, about 100-600 ng / It can be carried out at μL or a DNA segment or target polynucleotide concentration of about 100-500 ng / μL.

In some cases, the ligation reaction is about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3 hours, It can be carried out for longer than about 4 hours, about 5 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours or about 96 hours. .. In other cases, the ligation reaction is about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3 hours, about. It can be carried out for 4 hours, about 5 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours or less than about 96 hours. For example, the ligation reaction can be carried out for about 30 minutes to about 90 minutes. In some embodiments, conjugation of an adapter to a target polynucleotide produces a conjugated polynucleotide product with a 3'protrusion containing a nucleotide sequence from the adapter.

In some embodiments, after conjugating at least one adapter oligonucleotide to the target polynucleotide, the 3'end of one or more target polynucleotides uses one or more conjugated adapter oligonucleotides as a template. Is stretched. For example, with an adapter containing two hybridized oligonucleotides attached only to the 5'end of the target polynucleotide, at the same time as or after replacement of the unattached strand, using the attached strand of the adapter as a template. Allows unjunctioned 3'end elongation of the target. Both strands of the adapter containing two hybridized oligonucleotides are targeted to the polynucleotide so that the joined product has a 5'protrusion and the 5'protrusion can be used as a template to extend the complementary 3'end. Can be joined. As a further example, a hairpin adapter oligonucleotide can be attached to the 5'end of the target polynucleotide. In some embodiments, the 3'end of the extended target polynucleotide comprises one or more nucleotides from the adapter oligonucleotide. For target polynucleotides to which adapters are attached at both ends, extension can be performed at both 3'ends of double-stranded target polynucleotides with 5'protrusions. This 3'end extension or "fill-in" reaction produces a complementary sequence or "complement" to the adapter oligonucleotide template hybridizing to the template and thus fills the 5'protrusion to create a double-stranded sequence region. To make. If both ends of the double-stranded target polynucleotide have a 5'protrusion filled by extension of the 3'end of the complementary strand, the product is completely double-stranded. Elongation can be performed by any suitable polymerase known in the art, such as DNA polymerase, many of which are commercially available. The DNA polymerase can include DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity or DNA-dependent and RNA-dependent DNA polymerase activity. DNA polymerases can be thermostable or non-thermostable. Examples of DNA polymerases include, but are not limited to, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Sso polymerase, Poc polymerase, Pab. Polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerase, Platinum Taq polymerase, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Tru polymerase, Tac polymerase , Tne Polymerase, Tma Polymerase, Tih Polymerase, Tfi Polymerase, Klenow Fragment and variants thereof, modified products and derivatives. 3'end extension can be performed before or after pooling the target polynucleotide from an independent sample.

In certain embodiments, the present disclosure provides methods for enriching the target nucleic acid and analyzing the target nucleic acid. In some cases, the method of concentration is in the form of a solution system. In some cases, the target nucleic acid can be labeled with a labeling agent. In other cases, the target nucleic acid can be crosslinked to one or more associated molecules labeled with a labeling agent. Examples of labeling agents include, but are not limited to, biotin, polyhistidine tags and chemical tags (eg, alkyne and azide derivatives used in click chemistry). In addition, the labeled target nucleic acid can be captured and therefore enriched by using a scavenger. The scavenger is streptavidin and / or avidin, an antibody, a chemical moiety (eg, an alkyne, an azide), and any biological, chemical, physical or enzymatic agent used for affinity purification known in the art. obtain.

In some cases, immobilized or non-immobilized nucleic acid probes can be used to capture the target nucleic acid. For example, the target nucleic acid can be concentrated from the sample by hybridization to the probe on a solid support or in solution. In some examples, the sample can be a genomic sample. In some examples, the probe can be an amplicon. The amplicon can contain a default array. In addition, the hybridized target nucleic acid can be washed and / or eluted from the probe. The target nucleic acid can be a DNA, RNA, cDNA or mRNA molecule.

In some cases, the enrichment method can include contacting the probe with a sample containing the target nucleic acid and binding the target nucleic acid to the solid support. In some cases, the sample can be fragmented using chemical, physical or enzymatic methods to produce the target nucleic acid. In some cases, the probe can specifically hybridize to the target nucleic acid. In some cases, the target nucleic acid is about 50-5000, about 50-2000, about 100-2000, about 100-1000, about 200-1000, about 200-800 or about 300-800, about 300-600 or It can have an average size of about 400-600 nucleotide residues. The target nucleic acid can be further separated from the unbound nucleic acid in the sample. The solid support can be washed and / or eluted to give the enriched target nucleic acid. In some examples, the enrichment step can be repeated about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. For example, the enrichment step can be repeated about 1, 2 or 3 times.

In some cases, the enrichment method can include preparing a probe-derived amplicon, wherein the amplification probe is attached to a solid support. The solid support can include a nucleic acid probe immobilized on the support to capture a specific target nucleic acid from the sample. The probe-derived amplicon can hybridize to the target nucleic acid. After hybridization to the probe amplicon, the target nucleic acid in the sample is captured (eg, with a capture agent such as biotin, antibody, etc.), washed and / or by elution of the hybridized target nucleic acid from the captured probe. It can be concentrated (Fig. 4). The target nucleic acid sequence can be further amplified, for example using PCR methods, to create an amplified pool of concentrated PCR products.

In some cases, the solid support can be a microarray, slide, chip, microwell, column, tube, particle or bead. In some examples, the solid support can be coated with streptavidin and / or avidin. In another example, the solid support can be coated with an antibody. Further, the solid support can include glass, metal, ceramic or polymer materials. In some embodiments, the solid support can be a nucleic acid microarray (eg, a DNA microarray). In other embodiments, the solid support can be paramagnetic beads.

In some cases, the enrichment method can include digestion with a second restriction enzyme, self-ligation (eg, self-cyclization), and redigestion with the first restriction enzyme. In certain examples, only the ligation product will be linearized and will be available for adapter ligation and sequencing. In other cases, the ligation junction sequence itself can be used to concentrate based on hybridization using a bait probe complementary to the junction sequence.

In certain embodiments, the present disclosure provides a method of amplifying concentrated DNA. In some cases, the concentrated DNA is a read pair. Lead pairs can be obtained by the method of the present disclosure.

In some embodiments, one or more amplification and / or replication steps are used to prepare the library to be sequenced. Any amplification method known in the art can be used. Examples of amplification techniques that can be used are, but are not limited to, quantitative PCR, quantitative fluorescence PCR (QF-PCR), multiplex fluorescence PCR (MF-PCR), real-time PCR (RTPCR), single-cell PCR, limitation. Fragment length polymorphic PCR (PCR-RFLP), PCK-RFLPIRT-PCR-IRFLP, hot start PCR, nested PCR, in situ polonony PCR (polonony PCR), in situ rolling circle amplification (RCA), bridge PCR, ligation-mediated PCR , Qb Replicase Amplification, Inverse PCR, Picotiter PCR and Emulsion PCR. Other suitable amplification methods include ligase chain reaction (LCR), transcriptional amplification, autologous sustained sequence replication, selective amplification of target polynucleotide sequences, and consensus sequence primed polymerase chain reaction (consensus sequence primed polymerase chain reaction). There are CP-PCR, arbitrarily primed polymerase chain reaction (AP-PCR), degenerate oligonucleotide primed PCR (DOP-PCR) and nucleic acid sequence-based amplification (NABSA). Other amplification methods that can be used herein include those described in US Pat. Nos. 5,242,794, 5,494,810, 4,988,617, and 6,582,938.

In certain embodiments, the DNA molecule is dispensed into individual compartments and then PCR is used to amplify the DNA molecule. In some cases, one or more specific priming sequences in the amplification adapter are utilized for PCR amplification. The amplification adapter can be ligated to fragmented DNA molecules before or after dispensing into individual compartments. Polynucleotides containing amplification adapters with suitable priming sequences at both ends can be PCR amplified exponentially. For example, due to the incomplete ligation efficiency of amplification adapters containing priming sequences, polynucleotides with only one suitable priming sequence can only undergo linear amplification. Furthermore, if no adapter containing the appropriate priming sequence is ligated, the polynucleotide can be removed altogether from amplification, eg PCR amplification. In some embodiments, the number of PCR cycles varies from 10 to 30 cycles, but is as low as 9, 8, 7, 6, 5, 4, 3, 2, or less than 40, 45, It can be as many as 50, 55, 60 cycles. As a result, after PCR amplification, exponentially amplifyable fragments carrying an amplification adapter with the appropriate priming sequence have a very high concentration (1000) compared to linearly amplifyable or non-amplifiable fragments. Can be doubled or more). Compared to whole-genome amplification techniques (such as amplification with randomized primers or polysubstituted amplification using phi29 polymerase), the benefits of PCR are not limited to that, but more homogeneous relative sequence coverage, [each fragment is It can be copied at most once per cycle, and amplification is controlled by a thermal cycle program, resulting in a substantially lower rate of chimeric molecule formation than, for example, MDA (Lasken et al., 2007, BMC Biotechnology)] [. Chimera molecules pose a major challenge to accurate sequence assembly due to the presence of abiotic sequences in the assembly graph, which can lead to higher rates of misassembly or very vague and fragmented assembly. Compared to using a specific priming site with a specific sequence, the sequence-specific bias that can result from binding of randomized primers commonly used in MDA is reduced], the number of PCR cycles. High reproducibility of the amount of final amplified DNA product that can be controlled by selection, and high fidelity of replication by polymerases commonly used in PCR compared to common whole-genome amplification techniques known in the art. Can be mentioned.

In some embodiments, the fill-in reaction is continued or carried out as part of amplification of one or more target polynucleotides using the first and second primers, the first primer being the first primer. Contains a sequence capable of hybridizing to at least a portion of one or more complements of the adapter oligonucleotide of the second primer, and the second primer hybridizes to at least a portion of one or more complements of the second adapter oligonucleotide. Contains sequences that can be hybridized. Each of the first and second primers is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100 nucleotides or less or less. It can be of any suitable length, such as longer nucleotides, and any portion or all of it can be the corresponding target sequence (eg, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides or Can be complementary to lesser or longer nucleotides). For example, about 10-50 nucleotides can be complementary to the corresponding target sequence.

"Amplification" refers to any process in which the number of copies of a target sequence increases. In some cases, the replication reaction can make only one complementary copy / copy of the polynucleotide. Methods of primer-directed amplification of target polynucleotides are known in the art and include, but are not limited to, polymerase chain reaction (PCR) -based methods. Advantageous conditions for amplification of target sequences by PCR are known in the art and can be optimized at various steps in the process, targeting type, target concentration, amplified sequence length, target and / or. It depends on the characteristics of the element being reacted, such as the sequence of one or more primers, primer length, primer concentration, polymerase used, reaction volume, ratio of one or more elements to one or more other elements, etc. Some or all of them can be modified. Generally, PCR involves denaturation of the targeted target to be amplified (in the case of double strands), hybridization of one or more primers to the target, and extension of the primer with DNA polymerase, which is repeated to amplify the target sequence. Accompanied by (or "cycled") steps. Steps in this process can be optimized for a variety of outcomes such as increased yields, decreased fake product formation and / or increased or decreased specificity of primer annealing. Optimization methods are well known in the art and given in the course of the type or quantity of elements in the amplification reaction and / or the temperature at a particular step, the duration of a particular step and / or the number of cycles. Includes adjustment of step conditions.

In some embodiments, the amplification reaction comprises at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more cycles. be able to. In some examples, the amplification reaction can include at least about 20, 25, 30, 35 or 40 cycles. In some embodiments, the amplification reaction is about 5, 10, 15, 20, 25, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more cycles or less. The cycle can contain any number of steps, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more steps. Steps include, but are not limited to, 3'end extension (eg, adapter fill-in), primer annealing, primer extension and chain denaturation, including any temperature or temperature gradient suitable to achieve the purpose of a given step. be able to. Steps are about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 180, 240, 300, 360, 420, It may include any duration, including but not limited to 480, 540, 600, 1200, 1800 seconds or less, and unlimited time until manually interrupted. Any number of cycles containing different steps can be combined in any order. In some embodiments, the total number of cycles in the combination is about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 cycles or less. Or different cycles containing different steps are combined so that there are more. In some embodiments, amplification is performed after the fill-in reaction.

In some embodiments, the amplification reaction is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50. , 100, 200, 300, 400, 500, 600, 800, 1000 ng of target DNA molecules. In other embodiments, the amplification reaction is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100. , 200, 300, 400, 500, 600, 800, can be performed with target DNA molecules less than 1000 ng.

Amplification can be performed before or after pooling the target polynucleotide from an independent sample.

The methods of the present disclosure include determining the amount of amplifyable nucleic acid present in a sample. Any known method can be used to quantify the amplifyable nucleic acid, a typical method being a polymerase chain reaction (PCR), especially a quantitative polymerase chain reaction (qPCR). qPCR is a technique based on the polymerase chain reaction that uses it to amplify and simultaneously quantify targeted nucleic acid molecules. qPCR enables both detection and quantification of specific sequences in DNA samples (as an absolute number or relative amount of copies when standardized for input DNA or additional standardization genes). The procedure follows the general principles of the polymerase chain reaction, with the additional feature of quantifying the amplified DNA in real time as it accumulates after each amplification cycle in the reaction. For QPCR, for example, Kurnit et al. (US Pat. No. 6,033,854), Wang et al. (US Pat. Nos. 5,567,583 and 5,348,853), Ma et al. (The Journal of American Science, 2 (3), 2006), Heid et al. (Genome Research, pp. 986-994, 1996), Sambrook and Russell (Quantitative PCR, Cold Spring Harbor Protocols, 2006), and Higuchi US Pat. Nos. 6,171,785 and 5,994,056). These contents are incorporated herein by reference in their entirety.

Other methods of quantification include the use of fluorescent dyes that fall between double-stranded DNA and modified DNA oligonucleotide probes that fluoresce when hybridized with complementary DNA. Although these methods can be widely used, they are also particularly suitable for real-time PCR, as described in more detail as an example. In the first method, the DNA-binding dye binds to all double-stranded DNA (ds) in PCR, resulting in dye fluorescence. Therefore, the increase in DNA product during PCR results in an increase in fluorescence intensity, which is measured at each cycle and thus makes it possible to quantify the DNA concentration. The reaction is prepared in the same manner as a standard PCR reaction, and a fluorescent (ds) DNA dye is added. The reaction is performed in a thermocycler and after each cycle the fluorescence level is measured by the detector, the dye fluoresces only when it is bound to (ds) DNA (ie, the PCR product). The (ds) DNA concentration in PCR can be determined with reference to standard dilutions. As with other real-time PCR methods, the resulting value has no absolute unit associated with it. The comparison between the measured DNA / RNA sample and the standard dilution gives a fraction or ratio of the sample to the standard, allowing relative comparisons between different tissues or experimental conditions. It can be standardized for genes that are stably expressed to ensure accuracy in quantification and / or expression of the target gene. The number of copies of an unknown gene can be similarly standardized for a gene with a known number of copies.

The second method uses sequence-specific RNA or DNA-based probes to quantify only the DNA containing the probe sequences, so the use of reporter probes significantly increases specificity and provides some non-specificity. Allows quantification even in the presence of target DNA amplification. If all genes are amplified with comparable efficiency, this allows multiplexing, ie, assaying multiple genes in the same reaction using specific probes with different color labels. Become.

This method is commonly performed with DNA-based probes that have a fluorescent reporter (eg, 6-carboxyfluorescein) at one end of the probe and a fluorescent quencher (eg, 6-carboxytetramethylrhodamine) at the opposite end. ing. The proximity of the reporter to the quencher interferes with the detection of its fluorescence. Degradation of the probe by the 5'→ 3'exonuclease activity of a polymerase (eg, Taq polymerase) breaks the reporter-quenching substance proximity and thus emits unquenched fluorescence, which can be detected. .. The increase in products targeted by the reporter probe in each PCR cycle is brought about by a proportional increase in fluorescence due to probe degradation and reporter release. The reaction is prepared in the same manner as a standard PCR reaction and a reporter probe is added. When the reaction begins, both the probe and primer anneal to the DNA target during the PCR annealing phase. Polymerization of the new DNA strand is initiated from the primer, and once the polymerase reaches the probe, its 5'→ 3'exonuclease decomposes the probe, physically separating the fluorescent reporter from the quencher and fluorescing. Will increase. Fluorescence is detected and measured with a real-time PCR thermocycler and uses a geometric increase in fluorescence corresponding to the exponential increase in product to determine the threshold cycle in each reaction.

By plotting the fluorescence for the number of cycles on a logarithmic scale (so the exponentially increasing amount gives a straight line), the relative concentration of DNA present during the logarithmic phase of the reaction is determined. The threshold for detecting fluorescence above the background is determined. The cycle in which the fluorescence from the sample intersects the threshold is called the cycle threshold, C _t . During the logarithmic phase, the amount of DNA doubles with each cycle, so the relative amount of DNA can be calculated, for example, a sample with C _t that is 3 cycles earlier than another sample is 2 ³ = 8 times more. Has a mold. The results are then compared to a standard curve produced by real-time PCR of a known amount of serial dilution of nucleic acid (eg, stock solution, 1: 4, 1:16, 1:64) to nucleic acid (eg, RNA or The amount of DNA) is determined.

In certain embodiments, the qPCR reaction comprises a dual fluorophore technique utilizing fluorescence resonance energy transfer (FRET), such as the LIGHTCYCLER hybridization probe, where the two oligonucleotide probes are annealed to the amplicon. (See, for example, US Pat. No. 6,174,670). Oligonucleotides are designed to hybridize in the head-to-tail direction with fluorophore groups that are separated at distances that accommodate effective energy transfer. Another example of a labeled oligonucleotide structured to emit a signal when bound to a nucleic acid or integrated into an extension product is: SCORPIONS probe (eg Whitcombe et al., Nature Biotechnology 17:804). 807, 1999 and U.S. Pat. No. 6,326,145), Sunrise (or AMPLIFLOUR) primers (eg, Nazarenko et al., Nuc. Acids Res. 25: 2516-2521, 1997 and U.S. Pat. No. 6,117,635), and LUX primers. And molecular beacon probes (eg, Tyagi et al., Nature Biotechnology 14: 303-308, 1996 and US Pat. No. 5,989,823).

In other embodiments, the qPCR reaction uses the fluorescent Taqman method and an instrument capable of measuring fluorescence in real time (eg, ABI Prism 7700 Sequence Detector). The Taqman reaction uses a hybridization probe labeled with two different fluorochromes. One dye is a reporter dye (6-carboxyfluorescein) and the other is a quenching dye (6-carboxy-tetramethylrhodamine). When the probe is complete, fluorescence energy transfer occurs and the fluorescence emission of the reporter dye is absorbed by the quenching dye. During the extension phase of the PCR cycle, the fluorescent hybridization probe is cleaved by the 5'→ 3'nucleic acid degradation activity of the DNA polymerase. Cleavage of the probe no longer efficiently transfers the emission of the reporter dye to the quenching dye, resulting in an increase in the fluorescence emission spectrum of the reporter dye. Any nucleic acid quantification method, including real-time method or single point detection method, can be used to quantify the amount of nucleic acid in the sample. Detection is performed by several different methods (eg staining, hybridization with labeled probes, avidin-enzyme conjugate detection after incorporation with biotinylated primers, 32P labeled deoxynucleotide triphosphate to amplified segments such as dCTP or dATP). Incorporation of acid), and any other suitable detection method known in the art for nucleic acid quantification can be performed. The quantification may or may not include an amplification step.

In some embodiments, the present disclosure provides labels for identifying or quantifying linked DNA segments. In some cases, ligated DNA segments can be labeled to support downstream applications such as array hybridization. For example, ligated DNA segments can be labeled using random priming or nick translation.

Various labels (eg, reporters) can be used to label the nucleotide sequences described herein, including but not limited to amplification steps. Suitable labels include radionuclides, enzymes, fluorescence, chemiluminescence or color formers and ligands, cofactors, inhibitors, magnetic particles and the like. Examples of such markings are in US Pat. No. 3,817,837, US Pat. No. 3,850,752, US Pat. No. 3,939,350, US Pat. No. 3,996,345, US Pat. No. 4,277,437, US Pat. No. 4,275,149 and US Pat. No. 4,366,241. Included and incorporates in its entirety by reference.

Additional labels include, but are not limited to, β-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, β-glucuronidase, exoglucanase and glucoamylase. Fluorescent labels and specially synthesized fluorescent reagents with specific chemical properties can also be used. Various methods of measuring fluorescence are available. For example, some fluorescent labels exhibit excitation or changes in the emission spectrum, some exhibit resonance energy transfer, while one fluorescent reporter loses fluorescence while a second reporter increases fluorescence. It exhibits disappearance (quenching) or appearance of fluorescence, while some report rotational motion.

In addition, multiple amplifications can be pooled instead of increasing the number of amplification cycles per reaction in order to obtain sufficient material for labeling. Alternatively, labeled nucleotides can be incorporated into the final cycle of the amplification reaction, eg, 30 cycles of PCR (unlabeled) + 10 cycles of PCR (+ labeled).

In certain embodiments, the present disclosure provides probes capable of binding to ligated DNA segments. As used herein, the term "probe" refers to a molecule capable of hybridizing to another molecule of interest (eg, another oligonucleotide) (eg, is it naturally occurring, such as a purified restriction digest)? Or an oligonucleotide, whether synthetically, recombinantly or by PCR amplification). When the probes are oligonucleotides, they can be single-stranded or double-stranded. Probes are useful for the detection, identification and isolation of specific targets (eg, gene sequences). In some cases, the probe is detectable by any detection system including, but not limited to, enzymes (eg, ELISA and enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. Can be met with.

With respect to arrays and microarrays, the term "probe" is used to refer to any hybridizable material that is attached to an array for the purpose of detecting a nucleotide sequence that hybridizes to said probe. In some cases, the probe is at about 10 bp to 500 bp, about 10 bp to 250 bp, about 20 bp to 250 bp, about 20 bp to 200 bp, about 25 bp to 200 bp, about 25 bp to 100 bp, about 30 bp to 100 bp, or about 30 bp to 80 bp. possible. In some cases, the probe is about 10 bp, about 20 bp, about 30 bp, about 40 bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp, about 150 bp, about 200 bp, about 250 bp, It can be longer than about 300 bp, about 400 bp or about 500 bp. For example, the probe can be about 20 to about 50 bp in length. Examples and rationale for probe design can be found in WO95 / 11995, European Patent No. 717,113 and WO97 / 29212.

In some cases, one or more probes can be designed to hybridize near sites that are digested by restriction enzymes. For example, the probe is about 10 bp, about 20 bp, about 30 bp, about 40 bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp, about 150 bp, about 200 bp, about 250 bp, about 250 bp, about the restriction enzyme recognition site. It can be in the range of 300 bp, about 400 bp or about 500 bp.

In other cases, single and unique probes are about 10 bp, about 20 bp, about 30 bp, about 40 bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, on each side of the site digested by restriction enzymes. It can be designed in the range of about 100 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 400 bp or about 500 bp. Probes can be designed to hybridize to any side of the site that is digested by restriction enzymes. For example, a single probe can be used on each side of the primary restriction enzyme recognition site.

In additional cases, 2, 3, 4, 5, 6, 7, 8 or more probes can be designed on each side of the restriction enzyme recognition site, and then the same ligation is used using those probes. You can investigate the event. For example, two or three probes can be designed on each side of the restriction enzyme recognition site. In some examples, the use of multiple probes per primary restriction enzyme recognition site (eg, 2, 3, 4, 5, 6, 7 or 8 or more) is a problem with getting false negative results from individual probes. Can be useful in minimizing.

As used herein, the term "probe set" refers to a set or set of probes that can hybridize to one or more of the primary restriction enzyme recognition sites for a primary restriction enzyme in the genome.

In some cases, a set of probes can be sequence-complementary to nucleic acid sequences flanking one or more of the primary restriction enzyme recognition sites for restriction enzymes in genomic DNA. For example, a set of probes is about 10 bp to 500 bp, about 10 bp to 250 bp, about 20 bp to 250 bp, about 20 bp to 200 bp, about 25 bp to 200 bp, about 10 bp to 500 bp, about 10 bp to 250 bp, about 20 bp to 250 bp, about 25 bp to 200 bp, about nucleotides adjacent to one or more restriction enzyme recognition sites in genomic DNA. It can be complementary in sequence to 25 bp to 100 bp, about 30 bp to 100 bp, or about 30 bp to 80 bp. The set of probes can be complementary in sequence to one side (eg, either) or both sides of the restriction enzyme recognition site. Thus, the probe can be sequence-complementary to the nucleic acid sequences flanking each side of one or more of the primary restriction enzyme recognition sites in genomic DNA. In addition, a set of probes can be from one or more of the primary restriction enzyme recognition sites in genomic DNA: about 10 bp, about 20 bp, about 30 bp, about 40 bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp. , Can be complementary in sequence to nucleic acid sequences that are below about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 400 bp or about 500 bp.

In some cases, two or more probes can be designed to hybridize to sequences flanking one or more restriction enzyme recognition sites in genomic DNA. The probes can overlap or partially overlap.

A probe, an array of probes or a set of probes can be immobilized on a support. The support (eg, solid support) can be made of various materials such as glass, silica, plastic, nylon or nitrocellulose. The support is preferably rigid and has a planar surface. The support can have about 1-10,000,000 split seats. For example, the supports are about 10-10,000,000, about 10-5,000,000, about 100-5,000,000, about 100-4,000,000, about 1000-4,000,000, about 1000-3,000,000, about 10,000-3,000,000, about 10,000-2,000,000, about 100,000-2,000,000. Or it can have about 100,000 to 1,000,000 divided seats. The density of the divided loci can be at least about 10, about 100, about 1000, about 10,000, about 100,000 or about 1,000,000 divided loci in a range of 1 square centimeter. In some cases, each split locus can occupy> 95% with a single oligonucleotide. In other cases, each split locus can be occupied by a probe or a pooled mixture of probes. In a further case, some split loci are occupied by a probe or a pooled mixture of probes, and other split loci are occupied> 95% by a single oligonucleotide. Ru.

In some cases, the number of probes for a given nucleotide sequence on an array can be large excess for the DNA sample hybridized to such an array. For example, an array can have about 10, about 100, about 1000, about 10,000, about 100,000, about 1,000,000, about 10,000,000, or about 100,000,000 times the number of probes with respect to the amount of DNA in the input sample.

In some cases, an array can have about 10, about 100, about 1000, about 10,000, about 100,000, about 1,000,000, about 10,000,000, about 100,000,000 or about 1,000,000,000 probes.

An array of probes or a set of probes can be synthesized on a support in a stepwise manner, or can be combined in a pre-synthesized form. One method of synthesis is VLSIPS ™ (as described in US Pat. No. 5,143,854 and European Patent No. 476,014), which synthesizes oligonucleotide probes into dense, miniaturized arrays. With the use of light for. Mask design algorithms for reducing the number of synthetic cycles are described in US Pat. No. 5,571,639 and US Pat. No. 5,593,839. As described in European Patent No. 624,059, arrays can also be synthesized in a combinatorial fashion by feeding the monomers to the cells of the support via mechanically constrained channels. Arrays can also be synthesized by spotting reagents on the support using an inkjet printer (see, eg, European Patent No. 728,520).

In some embodiments, the present disclosure provides a method of hybridizing ligated DNA segments into an array. A "substrate" or "array" can be prepared either synthetically or biosynthetic, and is a deliberately produced collection of nucleic acids that can be screened for a variety of different forms of biological activity. (Eg, a library of soluble molecules, and a library of oligonucleotides linked to resin beads, silica chips or other solid supports). In addition, the term "array" includes a library of nucleic acids that can be prepared by spotting nucleic acids of essentially any length (eg, 1 to about 1000 nucleotide monomers in length) onto a substrate.

Array techniques and various related techniques and uses are usually described in many textbooks and documents. For example, Lemieux et al., 1998, Molecular Breeding 4, pp. 277-289, Schena and Davis, Parallel Analysis with Biological Chips. In PCR Methods Manual (edited by M. Innis, D. Gelfand, J. Sninsky), Schena and Davis, 1999, Genes, Genomes and Chips. In DNA Microarrays: A Practical Approach (edited by M. Schena), Oxford University Press, Oxford, UK, 1999), The Chipping Forecast (Nature Genetics special issue, 1999) January Supplement), Mark Schena (eds.), Microarray Biochip Technology, (Eaton Publishing), Cortes, 2000, The Scientist 14 [17]: 25 pages, Gwynn and Page, Microarray analysis, the next revolution in molecular biology, Science, August 6, 1999, and Eakins and Chu (1999) Trends in Biotechnology 17, pp. 217-218.

In general, by spatially separating the members of a library, any library can be arranged in an array in an orderly manner. Examples of libraries suitable for sequencing are live including nucleic acid libraries (library containing DNA, cDNA, oligonucleotides, etc.), peptides, polypeptides and protein libraries as well as ligand libraries and any other molecule. There is a rally.

The library can be immobilized or immobilized on a solid phase (eg, a solid substrate) to limit the diffusion and mixing of members. In some cases, a library of ligand-bound DNA can be prepared. In particular, the library can be immobilized on a substantially planar solid phase, including membranes and non-perforated substrates such as plastic and glass. In addition, libraries can be arranged for ease of indexing (ie, referencing or calling a particular member). In some examples, members of the library can be applied as spots in grid form. Common assay systems can be adapted for this purpose. For example, the array can be immobilized on the surface of the microplate with either multiple members in one well or a single member in each well. In addition, solid substrates such as nitrocellulose or nylon membranes (eg, membranes used in blotting experiments) can be membranes. Alternative substrates include glass or silica substrates. Thus, the library can be immobilized by any suitable method known in the art, for example, by charge interaction or by chemical bonding to the wall or bottom of the well or membrane surface. Other means of arranging and immobilizing, such as pipette manipulation, droplet contact, piezoelectric means, inkjet and bubble jet® technology, electrostatic coating, etc. can be used. In the case of silicon chips, photolithography can be used to arrange and secure the library on the chip.

Libraries can be arranged by being "spotted" onto a solid substrate, which can be done by hand or by using robotics to place the members accurately. Generally, an array can be described as a macroarray or a microarray, the difference being the size of the spot. Macroarrays can contain about 300 microns or larger spot sizes and can be easily imaged by existing gel and blot scanners. The spot size of microarrays can be less than 200 microns in diameter, and these arrays typically contain thousands of spots as well. Therefore, microarrays may require specialized robotics and imaging equipment, which may need to be specially ordered. Equipment is generally described in a review by Cortese, 2000, The Scientist 14 [11]:26.

Techniques for making immobilization libraries of DNA molecules are described in the art. Most of the conventional methods have usually described methods of synthesizing single-stranded nucleic acid molecule libraries using, for example, masking techniques that assemble different ordered sequences at different different positions on a solid substrate. U.S. Pat. No. 5,837,832 describes an improved method for making DNA arrays immobilized on silicon substrates based on extremely large integration techniques. In particular, U.S. Pat. No. 5,837,832 "tilings" to synthesize a particular set of probes in a spatially defined location on a substrate that can be used to create the immobilized DNA library of the present disclosure. It describes a strategy called. U.S. Pat. No. 5,837,832 also provides a reference for conventional techniques that can be used. In other cases, the array can also be assembled using photodeposited chemistry.

Arrays of peptides (or peptide mimetics) can also be synthesized on the surface in a manner that places each separate library member (eg, a unique peptide sequence) at a separate predefined location in the array. .. The identity of each library member is determined by its spatial location in the array. The location in the array where the binding interaction of a predetermined molecule (eg, target or probe) with the reactive library member occurs is determined, thereby identifying the sequence of the reactive library member based on the spatial location. For these methods, see US Pat. No. 5,143,854, WO 90/15070 and WO 92/10092, Fodor et al. (1991) Science, p. 251: 767, Dower and Fodor (1991) Ann. Rep. Med. Chem., 26:271. It is described on the page.

To aid detection, any readily detectable reporter, such as a label such as fluorescence, bioluminescence, phosphorescence, radioactive reporter, etc., can be used (as described above). Such reporters, their detection, binding to targets / probes, etc. are described elsewhere in this document. Labels for probes and targets are also disclosed in Shalon et al., 1996, Genome Res 6 (7): 639-45.

Examples of some commercially available microarray formats are given in Table 1 below (see also Marshall and Hodgson, 1998, Nature Biotechnology, 16 (1), pp. 27-31).

Signals can be detected to generate data from array-based assays to indicate the presence or absence of hybridization between the probe and the nucleotide sequence. In addition, direct and indirect labeling techniques can be utilized. For example, direct labeling integrates a fluorescent dye directly into a nucleotide sequence that hybridizes to an array-associated probe (eg, the dye is incorporated into the nucleotide sequence by enzymatic synthesis in the presence of labeled nucleotides or PCR primers). Direct labeling schemes can produce strong hybridization signals and are easy to implement, for example by using a family of fluorescent dyes with similar chemical structures and properties. Cyanine or alexa analogs can be utilized for multiple fluorescence comparison array analysis if the nucleic acid is directly labeled. In other embodiments, an indirect labeling scheme can be utilized to integrate the epitope into the nucleic acid before or after hybridization to the microarray probe. One or more staining procedures and reagents can be used to label the hybridized complex (eg, fluorescence by conjugation of a fluorescent molecule that binds to an epitope, thereby a dye molecule to an epitope of the hybridized species. You can get a signal).

In various embodiments, sequence information will be obtained from the nucleic acid molecules in the sample using the appropriate sequencing methods described herein or another method known in the art. Sequencing can be accomplished by classical Sanger sequencing methods well known in the art. Sequencing can also be achieved using high throughput systems, some of which detect the sequenced nucleotides immediately after or at the same time as they are integrated into the growth chain, ie in real time or substantially in real time. Allows to detect sequences. In some cases, high-throughput sequencing will generate at least 1,000, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 100,000 or at least 500,000 sequence reads per hour and sequence. Decision leads are at least about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 150, about 180, about 210, about 240, about 270, about 300, about 350, about It can be 400, about 450, about 500, about 600, about 700, about 800, about 900 or about 1000 bases.

In some embodiments, high-throughput sequencing is commercially available from Illumina's Genome Analyzer IIX, MiSeq personal sequencer, or HiSeq systems, such as those using HiSeq 2500, HiSeq 1500, HiSeq 2000 or HiSeq 1000 machines. Including the use of technology. These machines use synthetic terminator-based sequencing. These machines can perform 200,000,000,000 DNA reads or more in 8 days. Smaller systems can be utilized to run in 3, 2, 1 day or less time.

In some embodiments, high throughput sequencing involves the use of techniques available from ABI Solid System. This gene analysis platform enables large-scale parallel sequencing of clonally amplified DNA fragments linked to beads. The sequencing method is based on sequential ligation with dye-labeled oligonucleotides.

Next-generation sequencing can include ion semiconductor sequencing (eg, using Life Technologies technology (I on Torrent)). Ion semiconductor sequencing can take advantage of the fact that ions can be released when a nucleotide is integrated into a DNA strand. A high density array of micromeasurement wells can be formed to perform ion semiconductor sequencing. Each well can contain a single DNA template. Below the well may be an ion-sensitive layer and below the ion-sensitive layer may be an ion sensor. When a nucleotide is added to DNA, it can release H ⁺ , which can be measured as a change in pH. H ⁺ ions can be converted to voltage and recorded by a semiconductor sensor. The array chip can be flooded with nucleotides one after another. No scan, light or camera required. In some cases, the ION PROTON ™ sequencing device is used to sequence nucleic acids. In some cases, the ION PGM ™ sequencing device is used. Ion Torrent Personal Genome Machine (PGM). PGM can make 10,000,000 leads in 2 hours.

In some embodiments, high throughput sequencing involves the use of techniques available from Helicos BioSciences (Cambridge, Massachusetts), such as Single Molecule Sequencing by Synthesis (SMSS). SMSS is unique because it allows sequencing of the entire human genome within 24 hours. Finally, SMSS is described to some extent in US Patent Application Publication Nos. 2006/0024711, 2006/0024678, 2006/0012793, 2006/0012784, and 2005/0100932.

In some embodiments, high-throughput sequencing is a PicoTiter Plate device that includes a fiber optic plate that sends a chemiluminescent signal generated by a sequencing reaction recorded by a CCD camera in the instrument, such as 454 Lifesciences, Inc. (Branford, Branford, Includes the use of technology available from Connecticut). This use of optical fiber allows detection of at least 20,000,000 base pairs in 4.5 hours.

For methods of using fiber optic detection after bead amplification, see Marguile, M. et al., "Genome sequencing in microfabricated high-density pricolitre reactors", Nature, doi: 10.1038 / nature03959, and US Patent Application Publication No. 2002 / 0,012,930, It is described in 2003/0068629, 2003/0100102, 2003/0148344, 2004/0248161, 2005/0079510, 2005/0124022, and 2006/0078909.

In some embodiments, high-throughput sequencing is performed using a Clonal Single Molecule Array (Solexa, Inc.) or a time-of-synthesis decoding (SBS) utilizing a reversible terminator chemical reaction. Will be done. These technologies are described in US Patents 6,969,488, 6,897,023, 6,833,246, 6,787,308, and US Patent Application Publications 2004/0106110, 2003/0064398, 2003/0022207, and Constant, A., The Scientist 2003, 17 (13): 36, to some extent.

Next-generation sequencing techniques can include real-time technology [SMRT ™] by Pacific Biosciences. In SMRT, each of the four DNA bases can bind to one of four different fluorescent dyes. These dyes can be phosphate bonds. Single DNA polymerases can be immobilized with a single molecule template single-stranded DNA at the bottom of a zero-mode waveguide (ZMW). ZMW can be a confinement structure that allows observation of single nucleotide integration by DNA polymerase against a background of fluorescent nucleotides that can diffuse rapidly (microseconds) from ZMW. It can take a few milliseconds to integrate the nucleotide into the growth chain. During this time, the fluorescent label is excited, a fluorescent signal can be generated, and the fluorescent tag can be cleaved. The ZMW can be illuminated from the bottom. The light attenuated from the excitation rays can pass 20 to 30 nm below each ZMW. A microscope with a detection limit of 20 zepto liters ( ^10-21 liters) can be made. A small detection volume can provide a 1000x improvement in background noise reduction. Detection of the corresponding fluorescence of the dye can indicate which base was incorporated. The process can be repeated.

In some cases, next-generation sequencing is nanopore sequencing (see, eg, Soni GV and Meller A. (2007) Clin Chem 53: 1996-2001). The nanopores can be small holes as small as about 1 nanometer in diameter. By immersing the nanopores in a conductive liquid and applying a potential across the pores, a weak current due to the conduction of ions through the nanopores can be obtained. The amount of current flowing can be sensitive to the size of the nanopores. As the DNA molecule passes through the nanopores, each nucleotide on the DNA molecule can block the nanopores to a different extent. Therefore, the change in current that passes through the nanopores as the DNA molecule passes through the nanopores can represent a read-out of the DNA sequence. The nanopore sequencing technique can be from Oxford Nanopore Technologies, eg, the GridlON system. A single nanopore can be inserted into the polymer membrane across the top of the microwell. Each microwell can have an electrode for individual detection. Microwells with 100,000 or more microwells per chip (eg, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000 or 1,000,000 or more) can be produced in an array chip. A device (or node) can be used to analyze the chip. The data can be analyzed in real time. One or more devices can operate in time. The nanopores can be protein nanopores, such as protein α-hemolysin, heptamer protein pores. The nanopores can be solid nanopores, eg, 1 nanometer sized holes formed within a synthetic membrane (eg, SiN _x or SiO ₂ ). The nanopores can be complex pores (eg, accumulation of protein pores in a solid membrane). The nanopores can be nanopores with integrated sensors (eg tunnel electrode detectors, capacitive detectors or graphene nanogap or edge state detectors (eg Garaj et al. (2010) Nature, Vol. 67). , Doi: 10.1038 / nature09379)). Nanopores can be functionalized to analyze a particular type of molecule (eg, DNA, RNA or protein). Nanopore sequencing can include "chain sequencing", where the complete DNA polymer can pass through protein nanopores while being sequenced in real time as the DNA translocates the pores. Enzymes can separate strands of double-stranded DNA and impart strands through nanopores. DNA can have a hairpin at one end and the system can read both strands. In some cases, nanopore sequencing is "exonuclease sequencing," where individual nucleotides can be cleaved from the DNA strand by an advanced exonuclease, and the nucleotides pass through protein nanopores. can do. Nucleotides can transiently bind to molecules within the pores (eg, cyclodextran). The characteristic disruption of the current is used to identify the base.

GENIA nanopore sequencing technology can be used. The manipulated protein pores can be embedded in the lipid bilayer membrane. "Active control" technology is used to enable effective control of nanopore-membrane assembly and DNA movement through channels. In some cases, the nanopore sequencing technique is manufactured by NABsys. Genomic DNA can be fragmented into strands with an average length of about 100 kb. The 100 kb fragment can be single-stranded and then hybridized with the 6mer probe. Genome fragments with probes can traverse nanopores and create current-to-time tracking. Current tracking can provide probe positions on each genomic fragment. Genome fragments can be lined up to create a probe map for the genome. The process can be done in parallel with the probe library. A probe map of genome length can be generated for each probe. Errors can be corrected in a process called "Sequencing By Hybridization (mwSBH)". In some cases, the nanopore sequencing technique is manufactured by IBM / Roche. An electron beam can be used to create nanopore-sized openings in a microchip. An electric field can be used to attract or screw DNA through the nanopores. DNA transistor devices in nanopores can include nanometer-sized layers of alternating metal and dielectric. Separate charges in the DNA skeleton can be confined inside the DNA nanopores by an electric field. DNA sequences can be read by turning the gate voltage on and off.

Next-generation sequencing can include DNA nanoball sequencing (see, eg, Drmanac et al. (2010) Science 327: 78-81, as performed by Complete Genomics). DNA can be isolated, fragmented and size-selected. For example, DNA can be fragmented to an average length of about 500 bp (eg, by sonication). The adapter (Ad1) can be attached to the end of the fragment. Adapters can be used to hybridize to anchors for sequencing reactions. DNA with an adapter attached to each end can be PCR amplified. The adapter sequence can be modified so that the complementary single-stranded ends bind to each other to form circular DNA. The DNA can be methylated to protect it from cleavage by the IIS type restriction enzymes used in subsequent steps. The adapter (eg, the right adapter) can have a restriction recognition site, which can remain unmethylated. Unmethylated restriction recognition sites in the adapter can be recognized by restriction enzymes (eg, Acul), and the DNA can be cleaved by Acul at 13 bp to the right of the right adapter to form linear double-stranded DNA. can. The second round (Ad2) of the right and left adapters can be ligated to either end of the linear DNA, and any DNA bound to both adapters can be PCR amplified (eg, PCR). By). The Ad2 sequences can be modified to allow them to bind to each other and form circular DNA. DNA can be methylated, but restriction enzyme recognition sites can remain unmethylated at the left Ad1 adapter. Restriction enzymes (eg, Acul) can be applied and the DNA can be cleaved at 13 bp to the left of Ad1 to form linear DNA fragments. The third round (Ad3) of the right and left adapters can be ligated to the right and left surfaces of the linear DNA, and the resulting fragments can be PCR amplified. Adapters can be modified so that they can bind to each other and form circular DNA. A type III restriction enzyme (eg, EcoP15) can be added, which can cleave DNA at 26 bp on the left side of Ad3 and 26 bp on the right side of Ad2. This cleavage can remove large segments of the DNA and re-straighten the DNA. The fourth round (Ad4) of the right and left adapters can be ligated to DNA, the DNA can be amplified (eg, by PCR) and modified so that they bind to each other and the completed circular DNA. Form a mold.

Rolling circle replication (eg, using Phi29 DNA polymerase) can be used to amplify small pieces of DNA. The four adapter sequences can contain a hybridizable parindrome sequence, and the single strand can be folded over itself and average approximately 200-300 nanometers in diameter DNA nanoballs (DNB (DNB). Trademark)) can be formed. DNA nanoballs can be attached to microarrays (sequencing flow cells) (eg, by adsorption). The flow cell can be a silicon wafer coated with silicon dioxide, titanium and hexamethyldisilazane (HMDS) and a photoresist material. Sequencing can be performed by unlinked sequencing by ligating the DNA with a fluorescent probe. The color of the fluorescence at the inquired position can be visualized by a high resolution camera. The identity of the nucleotide sequences between the adapter sequences can be determined.

In some embodiments, high throughput sequencing can be performed using AnyDot.chips (Genovoxx, Germany). In particular, AnyDot.chips can enhance nucleotide fluorescence signal detection by 10x-50x. For information on AnyDot.chips and how to use them, see International Publication WO 02088382, WO 03020968, WO 03031947, WO 2005044836, PCT / EP 05/05657, PCT / EP 05/05655, and German Patent Application No. DE 101 49 786, DE 102 14 395, DE 103 56 837, DE 10 2004 009 704, DE 10 2004 025 696, DE 10 2004 025 746, DE 10 2004 025 694, DE 10 2004 025 695, DE 10 2004 025 744, DE 10 2004 025 It is described to some extent in 745 and DE 10 2005 012 301.

Other high-throughput sequencing systems include Venter, J. et al., Science, February 16, 2001, Adams, M. et al., Science, March 24, 2000, and MJ Levene et al., Science, 299: 682. ~ 686, January 2003, and US Patent Application Publication Nos. 2003/0044781 and 2006/0078937. Such an entire system comprises sequencing a target nucleic acid molecule having multiple bases by addition of bases over time by a polymerization reaction measured on the nucleic acid molecule, i.e., a template nucleic acid to be sequenced. The activity of nucleic acid polymerizing enzymes on the molecule is tracked in real time. The sequence can then be inferred by identifying which base is incorporated into the growth complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step of sequence base addition. The polymerase on the target nucleic acid molecule complex travels along the target nucleic acid molecule and is provided at a suitable position to extend the oligonucleotide primer at the active site. Multiple labeled nucleotide analogs are provided in the immediate vicinity of the active site, each of which is complementary to different nucleotides in the target nucleic acid sequence. The growing nucleic acid chain is extended by adding a nucleotide analog to the nucleic acid chain at the active site using a polymerase, and the nucleotide analog to be added is complementary to the nucleotide of the target nucleic acid at the active site. Nucleotide analogs added to oligonucleotide primers as a result of the polymerization step are identified. The steps of providing the labeled nucleotide analog, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated, whereby the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined. ..

In certain embodiments, the present disclosure further provides a kit comprising one or more components of the present disclosure. The kit can be used for any application apparent to those of skill in the art, including those described above. The kit can include, for example, multiple associated molecules, fixatives, restriction endonucleases, ligases and / or combinations thereof. In some cases, the associated molecule can be a protein, including, for example, histones. In some cases, the fixative can be formaldehyde or any other DNA cross-linking agent.

In some cases, the kit can further include multiple beads. The beads can be paramagnetic and / or coated with a scavenger. For example, beads can be coated with streptavidin and / or antibody.

In some cases, the kit can include adapter oligonucleotides and / or sequencing primers. In addition, the kit can include a device capable of amplifying read pairs using adapter oligonucleotides and / or sequencing primers.

In some cases, the kit comprises lysis buffer, ligation buffer (eg, dNTP, polymerase, polynucleotide kinase and / or ligase buffer, etc.) and PCR reagent (eg, dNTP, polymerase and / or PCR buffer, etc.). Can also include other reagents, but not limited to this,

The kit can also include instructions for using the components of the kit and / or for generating lead pairs.

The computer system 500 exemplified in FIG. 8 can be understood as a logical device capable of reading instructions from the medium 511 and / or the network port 505, the network port of which is optionally a server 509 having a fixed medium 512. Can be connected to. A system as shown in FIG. 8 can include any input device such as CPU 501, disk drive 503, keyboard 515 and / or mouse 516, as well as any monitor 507. Data communication can be realized for the server locally or remotely by the indicated communication medium. The communication medium can include any means of transmitting and / or receiving data. For example, the communication medium can be a network connection, a wireless connection or an internet connection. Such a connection can provide worldwide web communication. As illustrated in FIG. 8, it is assumed that the data associated with this disclosure may be transmitted over such networks or connections for reception and / or review by parties 522.

FIG. 9 is a block diagram illustrating a first architectural example of a computer system 100 that can be used in connection with the examples of embodiments of the present disclosure. As shown in FIG. 9, the example computer system can include a processor 102 for processing instructions. Unlimited examples of processors include Intel Xeon ™ processor, AMD Opteron ™ processor, Samsung 32-bit RISC ARM 1176JZ (F) -S v1.0 ™ processor, ARM Cortex-A8 Samsung S5 PC100 ™. There are processors, ARM Cortex-A8 Apple A4 ™ processors, Marvell PXA 930 ™ processors or functionally equivalent processors. Execution of multiple threads can be used for parallel processing. In some embodiments, whether in a single computer system within a cluster or distributed throughout the system over a network that includes multiple computers, mobile phones and / or personal data aids. , Multiple processors with multiple cores or processors can also be used.

As illustrated in FIG. 9, a fast cache 104 can be connected to or incorporated into the processor 102 to provide fast memory for instructions or data that are recently or frequently used by the processor 102. The processor 102 is connected to the north bridge 106 by the processor bus 108. The north bridge 106 is connected to the random access memory (RAM) 110 by the memory bus 112 and manages access to the RAM 110 by the processor 102. Northbridge 106 is also connected to Southbridge 114 by chipset bus 116. Southbridge 114 is then connected to peripheral bus 118. Peripheral buses can be, for example, PCI, PCI-X, PCI Express or other peripheral buses. Northbridges and southbridges, often referred to as processor chipsets, manage the movement of data between the processor and RAM and the peripheral components of peripheral bus 118. In some alternative architectures, the functionality of the northbridge can be built into the processor instead of using a separate northbridge chip.

In some embodiments, the system 100 may include an accelerator card 122 attached to the peripheral bus 118. Accelerators can include field programmable gate arrays (FPGAs) or other hardware to accelerate certain processes. For example, accelerators can be used to reconfigure adaptive data or evaluate algebraic expressions used in extended configuration processing.

The software and data are stored in external storage 124 and can be read into RAM 110 and / or cache 104 for use by the processor. System 100 is an operating system for managing system resources, (unlimited examples of operating systems are Linux®, Windows®, MACOS®, BlackBerry OS®, iOS®. ) And other functionally equivalent operating systems), as well as application software running on the operating system to manage data storage and optimization according to the examples of embodiments of the present disclosure.

In this example, system 100 is a network interface card (NIC) connected to a peripheral bus to obtain a network interface to external storage devices such as network attached storage (NAS) and other computer systems that can be used for distributed parallel processing. Also includes 120 and 121.

FIG. 10 is a schematic diagram showing a network 200 with a plurality of computer systems 202a and 202b, a plurality of mobile phones and personal data assisting devices 202c, and network attached storage (NAS) 204a and 204b. In an example of an embodiment, the systems 202a, 202b and 202c can manage data storage and optimize data access to the data stored in network attached storage (NAS) 204a and 204b. Mathematical models can be used for the data and evaluated using distributed parallel processing across computer systems 202a and 202b as well as mobile phones and personal data aid systems 202c. The computer systems 202a and 202b and the mobile phone and personal data auxiliary system 202c can also perform parallel processing to reconfigure the adaptive data of the data stored in the network attached storage (NAS) 204a and 204b. FIG. 10 is merely exemplary and various other computer architectures and systems can be used with the various embodiments of the present disclosure. For example, a blade server can be used for parallel processing. Processor blades can be connected to the back electrodes for parallel processing. The storage device can also be connected to the back electrode or as network attached storage (NAS) via a separate network interface.

In some examples of embodiments, the processor maintains separate memory space and can transmit data via a network interface, back electrode or other connector for parallel processing by another processor. In other embodiments, some or all of the processors may use the shared virtual address memory space.

FIG. 11 is a block diagram of a multiprocessor computer system 300 using a shared virtual address memory space according to an example of an embodiment. The system includes multiple processors 302a-f that can access the shared memory subsystem 304. The system incorporates multiple programmable hardware memory algorithm processors (MAPs) 306a-f into memory subsystem 304. Each MAP306a-f can include memories 308a-f and one or more field programmable gate array (FPGA) 310a-f. The MAP provides configurable functional units and can provide specific algorithms or parts of the algorithms to FPGA 310a-f for processing in close coordination with their respective processors. For example, MAP can be used to evaluate algebraic expressions for the data model and reconstruct the adaptive data in the examples of embodiments. In this example, each MAP is globally accessible by all of the processors of this purpose. In one configuration, each MAP can access the associated memory 308a-f using direct memory access (DMA), thereby being independent of and from their respective microprocessors 302a-f. It becomes possible to perform tasks asynchronously. In this configuration, the MAP can feed the results directly to another MAP for parallel execution of the pipeline and algorithms.

The computer architectures and systems described above are merely examples, and various other computers, mobile phones, and personal data auxiliary architectures and systems can be used as general purpose processors, coprocessors, FPGAs and other programmable logical devices, system-on-chip. Can be used with examples of embodiments that include (SOC), application specific integrated circuits (ASICs), and systems that use any combination of other processing and logic elements. In some embodiments, all or part of a computer system can be implemented within software or hardware. Embodiments of any variety of data storage media including random access memory, hard disks, flash memory, tape devices, disk arrays, network attached storage (NAS) and other localized or distributed data storage devices and systems. Can be used with the example of.

In an example of an embodiment, a computer system can be implemented using a software module that runs on any of the above or other computer architectures and systems. In other embodiments, the function of the system is firmware, a programmable logic device such as a field programmable gate array (FPGA) described in FIG. 11, a system on chip (SOC), an application specific integrated circuit (ASIC) or the like. Can be partially or completely implemented in the processing and logical elements of. For example, the configuration processor and the optimization program can be implemented by hardware acceleration using a hardware accelerator card such as the accelerator card 122 illustrated in FIG.

The following examples are for purposes of illustration only and do not limit this disclosure. They are representative of what can be used, while other procedures known to those of skill in the art can be used otherwise.

[Example 1]
How to produce chromatin in vitro

Two methods of reconstitution of chromatin are of particular interest: one method will use ATP-independent random deposition of histones on DNA, while the other will be periodic. Use an ATP-dependent assembly of nucleosomes. The present disclosure allows the use of any of the techniques by one or more of the methods disclosed herein. Examples of both methods of producing chromatin can be found in Lusser et al. ("Strategies for the reconstitution of chromatin", Nature Methods (2004), 1 (1): pp. 19-26), in which references are cited. Incorporated herein by reference in its entirety, including.

[Example 2]
Genome assembly using Hi-C based techniques

The genome from a human subject was fragmented into a pseudocontig with a size of 500 kb. Using a Hi-C-based method, multiple read pairs were generated by probing the physical layout of the chromosomes in living cells. Any number of Hi, including the method presented in Lieberman-Aiden et al. ("Comprehensive mapping of long range interactions reveals folding principles of the human genome", Science (2009), 326 (5950): 289-293). -C-based methods can be used to generate lead pairs, including the references contained therein, which are fully incorporated herein. Map read pairs to all false contigs and use pairs mapped to two separate false contigs to build an adjacency matrix based on the mapping data. Weighting at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99% of lead pairs by obtaining a mapping of the lead distance to the end of the fake contig. Mathematically incorporates a higher probability of short contacts than empirically known long contacts. For each sham contig, the adjacency matrix was then analyzed to determine the path through the sham contig by finding the single most adjacent sham contig, which path was determined to have the highest weight sum. .. By performing these methods, it was found that> 97% of all false contigs identified the correct adjacency. Additional experiments can be performed to test the effects of shorter contigs as well as alternative weighting and route finding schemes.

Alternatively, genomic assemblies that use Hi-C data can include computational methods that leverage genomic proximity signals in Hi-C datasets in the ultra-long-range scaffolds of the de novo genomic assembly. Examples of such calculations that can be used with the methods disclosed herein include the adjacent chromatin ligation method by Burton et al. (Nature Biotechnology 31: 111-1125 (2013)) and Kaplan et al. (Nature Biotechnology 31: 1143). There is a DNA trigonometric method according to (2013)), the reference thereof, and any citations therein are fully incorporated herein. Furthermore, it should be understood that these computational methods can be used in combination with the other genomic assembly methods presented herein.

For example, (a) clustering contigs into chromosome groups, (b) ordering contigs within one or more chromosome groups, and (c) assigning relative directions to individual contigs. Adjacent chromatin ligation methods based on Burton et al., Including, can be used in conjunction with the methods disclosed herein. For step (a), the contigs are grouped using hierarchical clustering. Each node initially represents one contig, and a graph is constructed in which each branch between the nodes has a weight equal to the number of Hi-C read pairs connecting the two contigs. Contigs are integrated together using hierarchical aggregated clustering by mean link distance and applied until the number of groups is reduced from separate chromosomes to the expected number (groups with two or more contigs). Only count). Repeated contigs (contigs standardized by the number of restricted fragment sites, whose average contigulation density including other contigs is twice greater than the average contigulation density) and contigs with few restricted fragment sites are not clustered. However, after clustering, if the average contigation density of the contigs in that group is four times greater than the average contigulation density of any other group, then each of these contigs is assigned to the group. For step (b), the graph is constructed like a clustering step, but with branch weights between nodes equal to the reciprocal of the number of Hi-C contigs between contigs, standardized by the number of restricted fragment sites per contig. Ru. Short contigs are excluded from this graph. The minimum spanning tree is calculated for this graph. The longest route of this tree, the "trunk", can be found. The spanning tree is then modified to lengthen the trunk by adding contigs adjacent to the trunk to the trunk in a manner that keeps the total branch weight empirically low. After seeing a long stem for each group, convert it to the complete order as follows: Remove the trunk from the spanning tree, leaving a set of "branches" containing all contigs that are not in the trunk. Reinsert these branches into the trunk, which is the first and longest branch at the selected insertion site, so as to maximize the number of connections between adjacent contigs while ordering. Short pieces are not reinserted, resulting in many of the small clustered contigs being omitted from the final assembly. For step (c), the orientation of each contig is determined in that order by considering the exact location of the Hi-C contiguous alignment on each contig. The likelihood of a Hi-C link connecting two reads at a genomic distance x is assumed to be approximately 1 / x per x ≧ about 100 Kb. A weighted directed acyclic graph (WDAG) is constructed that represents all possible ways to orient the contig in a given order. Each branch in WDAG corresponds to a pair of adjacent contigs in one of four possible directional combinations, and the branch weight is a log-likelihood that observes the set of Hi-C connection distances between the two contigs. It is set to degrees and it is assumed that they are directly adjacent in a given direction. For each contig, the quality score in that direction is calculated as follows. The log-likelihood of the set of Hi-C linkages observed between these contigs is found in and near its current direction. Then, the contig is inverted and the log-likelihood is recalculated. The first log-likelihood is guaranteed to be higher because the direction is calculated. The difference between the log-likelihoods is considered the quality score.

An alternative DNA triangulation similar to the method of Kaplan et al. Can be used in the method disclosed herein to assemble the genome from contigs and read pairs. DNA triangulation is based on the use of high-throughput genome-scale chromatin interaction data in vivo to infer the location of the genome. For DNA triangulation, the CTR pattern begins by dividing the genome into 100 kb bottles, each representing a large virtual contig, and calculating the average interaction frequency of each chromosome for each placed contig. Is quantified to. Interaction data for contigs with adjacent 1mbs on each side are excluded to assess long-range localization. Mean interaction frequency strongly separates intrachromosomal and interchromosomal interactions and is highly predictive of which chromosome the contig belongs to. Next, a simple multiclass model, the naive Bayes classifier, is trained to predict the chromosomes of each contig based on the mean interaction frequency of each chromosome. The assembled portion of the genome is used to fit a probabilistic single-parameter exponential decay model that describes the relationship between Hi-C interaction frequency and genomic distance (DDD pattern). Each time, the contig is removed from the chromosome with an adjacent region of 1 Mb on each side. The most probable position is then estimated for each contig based on the interaction profile and decay model. Prediction errors are quantified as the absolute value of the distance between the predicted position and the actual position.

Combining a long insertion library with DNA triangulation can further improve the predictability for each contig. Knowing the arrangement of chromosomes and the approximate location of each contig significantly reduces the complexity of calculating long insertion scaffolds, as each contig only needs to be paired with a nearby contig, thereby obscuring the contig. The mating can be eliminated and contigs located in distant regions of chromosomes or on different chromosomes can reduce assembly errors that are incorrectly mated.

[Example 3]
Haplotype fading method

Since the read pairs produced by the methods disclosed herein usually derive from intrachromosomal contacts, any read pair containing a heterozygous site will also retain information about its fading. This information can be used to perform reliable fading for short, medium and even longer (megabase) distances quickly and accurately. Experiments designed to fade data from one of a triad of 1000 genomes (one of the mother / father / child genomes) have definitely inferred fading. In addition, haplotype reconstruction using proximity ligation similar to Selvaraj et al. (Nature Biotechnology 31: 1111-1118 (2013)) can also be used with the haplotype fading method disclosed herein.

For example, haplotype reconstruction using proximity ligation-based methods can also be used in the methods disclosed herein for fading the genome. Haplotype reconstruction using a proximity ligation-based method combines proximity ligation and DNA sequencing with a probabilistic algorithm for haplotype assembly. First, proximity ligation sequencing is performed using chromosomal capture procedures such as the Hi-C procedure. These methods can capture DNA fragments from two distant genomic loci that wrap together in three-dimensional space. After the shotgun DNA sequencing of the resulting DNA library, the paired-end sequencing reads have an "insertion size" ranging from hundreds to tens of millions of base pairs. Thus, the short DNA fragments produced in the Hi-C experiment can produce small haplotype blocks, and the long fragments can ultimately ligate these small blocks. With sufficient sequencing coverage, this technique has the potential to concatenate variants into discontinuous blocks and assemble all such blocks into a single haplotype. This data is then combined with a probabilistic algorithm for haplotype assembly. The probabilistic algorithm utilizes a graph corresponding to overlapping sequence fragments in which nodes correspond to heterozygous variants and branches can concatenate variants. This graph may contain false branches due to sequencing errors or trans interactions. The maximum cut algorithm is then used to predict a conservative solution that best matches the haplotype information obtained by the set of input sequencing leads. Proximity ligation produces larger graphs than traditional genomic sequencing or mate pair sequencing, so the computation time and number of iterations can be modified to predict haplotypes with reasonable speed and accuracy. The data obtained can then be used to guide local fading using Beagle software and sequencing data from the Genome Project, thereby generating haplotypes across chromosomes with high resolution and accuracy.

[Example 4]
Metagenomic assembly method

Microorganisms are collected from the environment and fixed with a fixative such as formaldehyde to form crosslinks within the microbial cells. By using high-throughput sequencing, multiple microbial-derived contigs are generated. Multiple read pairs are generated by using Hi-C based techniques. Read pairs that map to different contigs indicate which contigs are from the same species.

[Example 5]
How to make a very long range of lead pairs (XLRP)

DNA is extracted to fragment sizes up to 150 kbp using a commercially available kit. Assemble DNA into reconstituted chromatin structures in vitro using a commercial kit from Activ Motif. Chromatin is biotinylated, fixed with formaldehyde, and immobilized on streptavidin beads. DNA fragments are digested with restriction enzymes and incubated overnight. The resulting sticky ends are filled with α-thio-dGTP and biotinylated dCTP to produce blunt ends. Ligase the blunt ends with T4 ligase. The reconstituted chromatin is digested with proteinase to recover the ligated DNA. DNA is extracted from the beads and subjected to exonuclease digestion to remove biotin from the unligated ends. The recovered DNA is sheared and the ends are filled with dNTP. The biotinylated fragment is purified by pull-down with streptavidin beads. In some cases, the adapter is ligated and the fragment is amplified with PCT for high throughput sequencing.

[Example 6]
How to make a high quality human genome assembly

The knowledge that this disclosure can generate read pairs across significant genomic distances allows us to test the use of this information for genomic assembly. The present disclosure can significantly improve the ligation of de novo assemblies, potentially down to chromosomal length scaffolds. Determination can be made as to how complete the assembly can be made using the present disclosure and how much data will be required. To assess the effectiveness of this method in producing useful data for assembly, standard Illumina shotgun and XLRP libraries can be assembled and sequenced. In some cases, one lane of Illumina HiSeq data is used for each of the standard shotgun and XLRP libraries. The data generated from each method is tested and compared to various existing assemblers. The new assembler is also described, optionally, to be tailored specifically to the unique data produced by the present disclosure. Optionally, a well-characterized human sample is used to prepare a reference for comparison to the assembly made by the method, thereby determining the accuracy and completeness of the method. The findings obtained from previous analyses will be used to create assemblers to enhance the effective and effective use of XLRP and shotgun data. A December 2002 mouse genome draft or better quality genome assembly is generated using the methods described herein.

One of the samples that can be used for this analysis is NA12878. The DNA of the sample cells is extracted using a variety of published techniques designed to maximize DNA fragment length. A standard Illumina TruSeq shotgun library and XLRP library are assembled respectively. A single lane 2 × 150 bp sequence of HiSeq is obtained for each library, which can produce approximately 150,000,000 read pairs per library. Shotgun data is assembled into contigs using whole-genome assembly algorithms. Examples of such algorithms are described in Meraculous or Simpson et al. (Genome research 22 (3): 549-56 (2012)) described in Chapman et al. (PLOS ONE 6 (8): e2350 (2011)). There is SGA. XLRP library reads are aligned to the contig created by the first assembly. Alignment is used to further connect the contigs. Once the effectiveness of the XLRP library on connecting contigs can be seen, extend the Meraculous assembly to include the shotgun and XLRP library in a single assembly process at the same time. Meraculous provides a strong foundation for assembler. Optionally, an all-in-one assembler is made to meet the specific needs of the present disclosure. The human genome assembled by the present disclosure is compared to any known sequence to assess the quality of the genome assembly.

[Example 7]
How to Fad Heterozygous SNPs in Human Samples with High Precision from Small Data Sets

In one experiment, approximately 44% of the heterozygous variants in the test human sample dataset are fading. All or almost all fading variants within a one-lead length distance of the restriction site are captured. By using in silico analysis, more variants for fading can be captured by using longer read lengths and by using one or more combinations of restriction enzymes for digestion. The use of a combination of restriction enzymes with different restriction sites increases the proportion of genomes (and thus heterozygous sites) within one of the two restriction sites involved in each read pair. In silico analysis shows that the methods disclosed are capable of fading over 95% of known heterozygous positions using various combinations of two restriction enzymes. Additional enzymes as well as longer read lengths further increase the fraction of the observed and faded heterozygous sites to full coverage and fading.

Calculate the coverage of heterozygous sites that can be achieved with various combinations of the two restriction enzymes. The top three combinations for heterozygous sites in lead proximity are tested in this procedure. For each of these combinations, an XLRP library is created and sequenced. The resulting reads are aligned with the human reference genome and compared to the known haplotypes of the sample to determine the accuracy of the procedure. Up to 90% or more of the heterozygous SNPs in human samples are faded with a high accuracy of 99% or more using data from only one lane of Illumina HiSeq. In addition, further variants are captured by increasing the read length up to 300 bp. The lead area around the observable restricted area is effectively doubled. Implement additional restriction enzyme combinations to increase coverage and accuracy.

[Example 8]
Extraction and effects of high molecular weight DNA

DNA of 150 kbp or less was extracted with a commercially available kit. Figure 7 demonstrates that XLRP libraries can be generated from captured read pairs up to the maximum fragment length of the extracted DNA. Therefore, the methods disclosed herein can be expected to have the ability to generate read pairs from longer stretches of DNA. There are many well-developed processes for recovering high molecular weight DNA, and these methods can be used in conjunction with the methods or procedures disclosed herein. Extraction methods that produce large fragment length DNA can be used to create XLRP libraries from these fragments and evaluate the resulting read pairs. For example, DNA with a large molecular weight can be found in (1) Teague et al. (Proc. Nat. Acad. Sci. USA 107 (24): 10848-53 (2010)) or Zhou et al. (PLOS Genetics, 5 (11) ,: e1000711. References by gentle lysis of cells by page (2009)) and by agarose gel plugs by (2) Wing et al. (The Plant Journal: for Cell and Molecular Biology 4 (5): 893-8 (1993)). Can be extracted by using the Aurora System manufactured by Boreal Genomics, which is fully incorporated herein by reference, including any of the references contained therein. These methods are capable of producing long DNA fragments that exceed the DNA required for conventional next-generation sequencing methods, but any other suitable method known in the art, with similar results. It can be substituted to achieve it. The Aurora System provides unique results and can separate and concentrate DNA from tissues or other specimens up to and over 1 megabase in length. Using each of these methods, DNA extractions are prepared starting with a single GM12878 cell culture to control possible differences at the sample level. Fragment size distribution can be evaluated by pulsed-field gel electrophoresis by Herschleb et al. (Nature Protocols 2 (3): 677-84 (2007)). The methods described above can be used to extract very large DNA stretches and use them to assemble an XLRP library. The XLRP library is then sequenced and sorted. Read data obtained by comparing the genomic distance between read pairs to the fragment size observed from the gel is analyzed.

[Example 9]
Reduce read pairs from undesired genomic regions

RNA complementary to the undesired genomic region is produced by in vitro transcription and added to the reconstituted chromatin prior to cross-linking. RNA binding reduces cross-linking efficiency in these regions as the supplemented RNA binds to one or more undesired genomic regions. Thereby, the abundance of DNA from these regions in the crosslinked complex is reduced. Reconstructed chromatin is biotinylated, immobilized and used as described above. In some cases, RNA is designed to target repetitive regions in the genome.

[Example 10]
Increases lead pairs from the desired chromatin region

DNA from the desired chromatin region is generated in double-stranded form for gene assembly or haplotyping. Therefore, the appearance of DNA from undesired regions is reduced. Double-stranded DNA from the desired chromatin region is produced in such region by primers that tile at multiple kilobase intervals. In another implementation of the method, tiling intervals are varied to address desired regions of different sizes with the desired replication efficiency. By optionally melting the DNA, the primer binding site across the desired region is brought into contact with the primer. New strands of DNA are synthesized using tiled primers. Targeting these regions with, for example, single-stranded DNA-specific endonucleases reduces or eliminates unwanted regions. The remaining desired region can be optionally amplified. The prepared sample is subjected to the sequencing library preparation method described elsewhere herein. In some implementations, lead pairs spanning the distance to the length of each desired chromatin region are generated from each of such desired chromatin regions.

Preferred embodiments of the present disclosure have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided as just one example. A number of modifications, changes and replacements will be immediately conceivable to those of skill in the art without departing from this disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein are available in practicing the present disclosure. The following claims define the scope of this disclosure, thereby covering the methods and structures within the scope of these claims and their equivalents. The present invention includes the following embodiments.
[1] A method of genome assembly,
Steps to generate multiple contigs and
Steps to generate multiple read pairs from data produced by probing the physical layout of chromosomes, chromatin or reconstituted chromatin,
A step of mapping or assembling the plurality of read pairs to the plurality of contigs,
The step of constructing a contig adjacency matrix using the read mapping or assembly data,
With the step of analyzing the adjacency matrix to determine the route through the contig, which represents its order and / or direction with respect to the genome.
How to include.
[2] The plurality of contigs
With the steps of fragmenting a long stretch of DNA of interest into random pieces of uncertain size,
Steps to sequence the fragment using a high-throughput sequencing method to generate multiple sequencing reads,
With the step of assembling the sequencing read to form multiple contigs
The method according to embodiment 1, which is generated by using a shotgun sequencing method comprising.
[3] The first or second embodiment, wherein the plurality of read pairs are generated by probing the physical layout of a chromosome, chromatin or reconstituted chromatin using a Hi-C based technique. the method of.
[4] The technique based on Hi-C is
Steps to crosslink chromosomes, chromatin or reconstituted chromatin with a fixative to form DNA-protein crosslinks,
A step of cleaving the crosslinked DNA-protein with one or more restriction enzymes to generate multiple DNA-protein complexes containing sticky ends.
A step of filling the sticky end with a nucleotide containing one or more markers to create a blunt end to be ligated together.
The step of fragmenting the plurality of DNA-protein complexes into fragments,
With the step of pulling down the junction containing the fragment by using one or more of the markers,
With the step of sequencing the junction containing the fragment using a high throughput sequencing method to generate multiple read pairs.
The method according to the third embodiment.
[5] The method of any of the embodiments, wherein the plurality of read pairs are generated by probing the physical layout of a chromosome or chromatin isolated from cultured cells or primary tissue.
[6] Probing the physical layout of the reconstituted chromatin formed by complexing the naked DNA obtained from one or more target samples with isolated histones by the plurality of read pairs. The method according to any one of embodiments 1 to 4, which is generated by.
[7] In the case of the plurality of lead pairs, at least about 80% of the lead pairs are weighted by obtaining a mapping of the distance of the leads to the end of the contig, and the short contact is higher than the long contact. The method according to any of the above embodiments, which incorporates probabilities.
[8] The method of any of the embodiments, wherein the adjacency matrix is rescaled to reduce the weight of many contacts on the contig representing an indiscriminate region of the genome.
[9] The method of embodiment 8, wherein the indiscriminate region of the genome comprises one or more conservative binding sites for one or more agents that regulate chromatin scaffold interactions.
[10] The method of embodiment 9, wherein the one or more agents comprises a transcriptional repressor CTCF.
[11] The human subject's chromosome or chromatin, which provides the genomic assembly of the human subject, wherein the plurality of contigs are generated from the human subject's DNA and the plurality of read pairs are made from the subject's naked DNA. Alternatively, the method according to any of the above embodiments, which is produced by using reconstituted chromatin.
[12] A method for determining haplotype fading, comprising the method according to any of the above embodiments.
The step of identifying one or more heterozygous sites in the plurality of read pairs,
Steps to identify a lead pair containing a pair of heterozygous sites
The method by which fading data for allelic variants can be determined by the identification of the pair of heterozygous sites.
[13] A method of metagenomic assembly, comprising the method according to embodiment 1, wherein the plurality of lead pairs are composed of the plurality of lead pairs.
Steps to collect microorganisms from the environment,
With the step of adding a fixative to form crosslinks within each microbial cell
Lead pairs determined by probing the physical layout of multiple microbial chromosomes using a modified Hi-C-based method, including, and mapped to different contigs, which contigs are from the same species. How to show.
[14] The method of embodiment 13, wherein the fixative is formaldehyde.
[15] A method of assembling multiple contigs generated from a single DNA molecule.
The step of generating multiple read pairs from the single DNA molecule,
With the step of assembling the contig using a lead pair
A method in which at least 1% of the read pairs span a distance of at least 50 kB on the single DNA molecule and the read pairs are generated within 14 days.
[16] The method of embodiment 15, wherein at least 10% of the read pairs span a distance of at least 50 kB on the single DNA molecule.
[17] The method of embodiment 15, wherein at least 1% of the read pairs span a distance of at least 100 kB on the single DNA molecule.
[18] The method of any of embodiments 15-17, wherein the lead pair is generated within 7 days.
[19] A method of assembling multiple contigs derived from a single DNA molecule.
In vitro, the step of generating multiple read pairs from the single DNA molecule,
With the step of assembling the contig using the lead pair
A method in which at least 1% of the read pairs span a distance of at least 30 kB on the single DNA molecule.
[20] The method of embodiment 19, wherein at least 10% of the read pairs span a distance of at least 30 kB on the single DNA molecule.
[21] The method of embodiment 20, wherein at least 1% of the read pairs span a distance of at least 50 kB on the single DNA molecule.
[22] A haplotype fading method,
Steps to generate multiple read pairs from a single DNA molecule,
The haplotype fading comprises at least 1% of the read pairs spanning a distance of at least 50 kB on the single DNA molecule, comprising assembling multiple contigs of the DNA molecule using the read pairs. A method implemented with an accuracy of over 70%.
[23] The method of embodiment 22, wherein at least 10% of the read pairs span a distance of at least 50 kB on the single DNA molecule.
[24] The method of embodiment 22, wherein at least 1% of the read pairs span a distance of at least 100 kB on the single DNA molecule.
[25] The method of any of embodiments 22-24, wherein the haplotype fading is performed with an accuracy greater than 90%.
[26] A haplotype fading method,
Steps to generate multiple read pairs from a single DNA molecule in vitro,
The haplotype fading comprises at least 1% of the read pairs spanning a distance of at least 30 kB on the single DNA molecule, comprising assembling multiple contigs of the DNA molecule using the read pairs. A method implemented with an accuracy of over 70%.
[27] The method of embodiment 26, wherein at least 10% of the read pairs span a distance of at least 30 kB on the single DNA molecule.
[28] The method of embodiment 26, wherein at least 1% of the read pairs span a distance of at least 50 kB on the single DNA molecule.
[29] The method of any of embodiments 26-28, wherein the haplotype fading is performed with an accuracy greater than 90%.
[30] A method of in vitro haplotype fading in which haplotype fading is performed with an accuracy of over 70%.
[31] A method of generating a first read pair from a first DNA molecule.
(a) A step of cross-linking the first DNA molecule in vitro, wherein the first DNA molecule contains a first DNA segment and a second DNA segment.
(b) A step of connecting the first DNA segment to the second DNA segment to form a linked DNA segment.
(c) With the step of sequencing the linked DNA segment and thereby obtaining a first read pair.
How to include.
[32] The method of embodiment 31, wherein the plurality of associated molecules are crosslinked to the first DNA molecule.
[33] The method of embodiment 32, wherein the associated molecule comprises an amino acid.
[34] The method of embodiment 33, wherein the associated molecule is a peptide or protein.
[35] The method of any of embodiments 31-34, wherein the first DNA molecule is crosslinked with a fixative.
[36] The method of embodiment 35, wherein the fixative is formaldehyde.
[37] The method of any of embodiments 31-36, wherein the first DNA segment and the second DNA segment are generated by cleaving the first DNA molecule.
[38] The method of any of embodiments 31-37, further comprising assembling a plurality of contigs of the first DNA molecule using the first read pair.
[39] Embodiments 31-38, wherein each of the first and second DNA segments is attached to at least one affinity label and the linked DNA segment is captured using the affinity label. The method described in either.
[40] (a) A step of providing a plurality of associated molecules to at least a second DNA molecule.
(b) A step of cross-linking the associated molecule to the second DNA molecule, thereby forming a second complex in vitro.
(c) A step of cleaving the second complex to generate a third DNA segment and a fourth segment.
(d) A step of linking the third DNA segment with the fourth DNA segment to form a second linked DNA segment.
(e) With the step of sequencing the second linked DNA segment and thereby obtaining a second read pair.
31. The method of embodiment 31.
[41] The method of embodiment 40, wherein less than 40% of the DNA segment from said DNA molecule is linked to a DNA segment from any other DNA molecule.
[42] The method of embodiment 40, wherein less than 20% of the DNA segment from said DNA molecule is linked to a DNA segment from any other DNA molecule.
[43] A method of generating a first read pair from a first DNA molecule containing a predetermined sequence.
(a) A step of providing one or more DNA-binding molecules to the first DNA molecule, wherein the one or more DNA-binding molecules bind to the predetermined sequence.
(b) A step of cross-linking the first DNA molecule in vitro, wherein the first DNA molecule contains a first DNA segment and a second DNA segment.
(c) A step of linking the first DNA segment to the second DNA segment, thereby forming a first linked DNA segment.
(d) With the step of sequencing the first linked DNA segment, thereby obtaining the first read pair.
A method in which the probability that the predetermined sequence appears in the read pair is affected by the binding of the DNA-binding molecule to the predetermined sequence.
[44] The method of embodiment 43, wherein the DNA binding molecule is a nucleic acid capable of hybridizing to the predetermined sequence.
[45] The method of embodiment 44, wherein the nucleic acid is RNA.
[46] The method of embodiment 44, wherein the nucleic acid is DNA.
[47] The method of embodiment 43, wherein the DNA binding molecule is a small molecule.
[48] The method of embodiment 47, wherein the small molecule binds to the predetermined sequence with a binding affinity of less than 100 μM.
[49] The method of embodiment 47, wherein the small molecule binds to the predetermined sequence with a binding affinity of less than 1 μM.
[50] The method of any of embodiments 43-49, wherein the DNA-binding molecule is immobilized on a surface or a solid support.
[51] The method of embodiment 43, wherein the probability that the predetermined sequence will appear in the read pair is reduced.
[52] The method of embodiment 43, wherein the probability that the predetermined sequence will appear in the read pair is increased.
[53] An in vitro library containing a plurality of read pairs, each containing at least a first sequence element and a second sequence element, wherein the first and second sequence elements are derived from a single DNA molecule. , A library containing first and second sequence elements in which at least 1% of the read pairs are separated by at least 50 kB on the single DNA molecule.
[54] The in vitro library according to embodiment 53, wherein at least 10% of the read pair comprises first and second sequence elements separated by at least 50 kB on the single DNA molecule.
[55] The in vitro library according to embodiment 54, wherein at least 1% of the read pair comprises first and second sequence elements separated by at least 100 kB on the single DNA molecule.
[56] The in vitro library according to any of embodiments 53-55, wherein less than 20% of the read pairs contain one or more predetermined sequences.
[57] The in vitro library according to embodiment 56, wherein less than 10% of the read pairs contain one or more predetermined sequences.
[58] The in vitro library according to embodiment 57, wherein less than 5% of the read pairs contain one or more predetermined sequences.
[59] The in vitro library according to any of embodiments 56-58, wherein the defined sequence is determined by one or more nucleic acids or small molecules capable of hybridizing to the defined sequence.
[60] The in vitro library according to embodiment 59, wherein the one or more nucleic acids are RNA.
[61] The in vitro library according to embodiment 59, wherein the one or more nucleic acids are DNA.
[62] The in vitro library according to any of embodiments 59-61, wherein the one or more nucleic acids are immobilized on a surface or a solid support.
[63] The in vitro library according to embodiment 59, wherein the predetermined sequence is determined by one or more small molecules.
[64] The in vitro library according to embodiment 63, wherein the one or more small molecules bind to the predetermined sequence with a binding affinity of less than 100 μM.
[65] The in vitro library according to embodiment 63, wherein the one or more small molecules bind to the predetermined sequence with a binding affinity of less than 1 μM.
[66] A composition comprising a DNA fragment and a plurality of associated molecules, wherein the associated molecule is crosslinked to the DNA fragment in an in vitro complex, and the in vitro complex is immobilized on a solid support. The composition that has been made.
[67] A composition comprising a DNA fragment, a plurality of associated molecules and a DNA binding molecule, wherein the DNA binding molecule is bound to a predetermined sequence of the DNA fragment and the associated molecule is attached to the DNA fragment. The crosslinked composition.
[68] The composition according to embodiment 67, wherein the DNA-binding molecule is a nucleic acid capable of hybridizing to the predetermined sequence.
[69] The composition according to embodiment 68, wherein the nucleic acid is RNA.
[70] The composition according to embodiment 68, wherein the nucleic acid is DNA.
[71] The composition according to any of embodiments 68-70, wherein the nucleic acid is immobilized on a surface or a solid support.
[72] The composition according to embodiment 67, wherein the DNA-binding molecule is a small molecule.
[73] The composition according to embodiment 72, wherein the small molecule binds to the predetermined sequence with a binding affinity of less than 100 μM.
[74] The composition according to embodiment 72, wherein the small molecule binds to the predetermined sequence with a binding affinity of less than 1 μM.

Claims (20)

Contacting the sample with a fixative , wherein the sample comprises a nucleic acid molecule complexed to at least one nucleic acid binding protein.
Cleaving the nucleic acid into multiple segments, including at least the first segment and the second segment.
Connecting the first segment and the second segment at a joint,
Obtaining at least a portion of the sequence on each side of the junction to generate the first read pair,
Mapping the first read pair to a set of contigs and determining a route through the set of contigs that represents the direction and / or order with respect to the genome.
How to include. Excluding the first read pair from the contig assembly analysis because at least a portion of the sequence on each side of the junction maps to a common contig, and using a second read pair against the genome. Determining the route through the contig, which represents the direction and / or order.
The method according to claim 1. The method of claim 1, comprising contacting the sample with an antibody prior to contacting the sample with a fixative . The method of claim 1, comprising contacting the sample with a fixative followed by contacting the sample with an antibody. The method of claim 1, wherein cleaving the nucleic acid into at least a first segment and a second segment comprises cleaving the nucleic acid with at least one restriction enzyme. The method of claim 1, wherein cleaving the nucleic acid into at least a first segment and a second segment comprises shearing the nucleic acid. The method of claim 1, wherein cleaving the nucleic acid into at least a first segment and a second segment comprises sonicating the nucleic acid. The method of claim 1, wherein contacting the sample with a fixative comprises irradiating with ultraviolet light. The method of claim 1, wherein contacting the sample with a fixative comprises contacting the sample with a chemical fixative. The method of claim 9, wherein the chemical fixative comprises formaldehyde. The method of claim 9, wherein the chemical fixative comprises psoralen. The method of claim 1, wherein the at least one nucleic acid binding protein comprises a natural chromatin component. The method of claim 1, wherein the at least one nucleic acid binding protein comprises an externally sourced histone. The method of claim 1, wherein the ligation comprises filling the sticky end with at least some biotin-labeled nucleotide and ligating the blunt end. The method of claim 1, wherein the route through a set of contigs representing orientation and / or order with respect to the genome is determined such that each contig is recalled exactly once. The method of claim 1, wherein determining a route through a set of contigs representing the direction and / or order with respect to the genome reduces the weight of the contigs representing indiscriminate regions of the genome. The method of claim 1, wherein the set of contigs is generated by a shotgun sequencing method. The method of claim 1, wherein determining a pathway through a set of contigs representing orientation and / or order with respect to the genome comprises haplotype fading of the set of contigs. Haplotype fading of a set of contigs involves identifying one or more heterozygous sites in multiple read pair sequences, where fading data for allelic variants contains reads containing pairs of heterozygous sites. 18. The method of claim 18, as determined by identifying a pair. Claim 1 excludes read-pair sequences that map to a common contig from analysis, thereby using read pairs that map to different contigs to determine a route through a set of contigs that represent orientation and / or order with respect to the genome. The method described in.

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